Management of pathogenic lawsonia

ABSTRACT

The present invention relates to an animal feed or an animal feed additive comprising  Bacillus  strains which improve the health and performance of production animals, and the use of such.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Application No.16/965,866, filed Jul. 29, 2020 (now pending), which is a 35 U.S.C. 371national application of international application no. PCT/US2019/016271,filed Feb. 1, 2019, and published as International Patent ApplicationPublication WO2019/152791 on Aug. 8, 2019, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. Provisional Application No.62/625,549, filed Feb. 2, 2018, the contents of each of which are fullyincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference. The name of the filecontaining the Sequence Listing is SQ.XML, which was created on Sep. 20,2022 and has 21.4 KB.

Index to sequence listing:

-   SEQ ID NO: 1 is 16S rDNA of DSM 32559-   SEQ ID NO: 2 is 16S rDNA of DSM 32560-   SEQ ID NO: 3 is 16S rDNA of DSM 32561-   SEQ ID NO: 4 is 16S rDNA of DSM 32563-   SEQ ID NO: 5 to SEQ ID NO: 10: PCR and sequencing primers-   SEQ ID NO: 11 is 16S rDNA of O52YZ5-   SEQ ID NO: 12 is 16S rDNA of O22FHD.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference. Forcomplete information see last paragraph of the description.

FIELD OF THE INVENTION

The present invention relates to an animal feed or an animal feedadditive comprising Bacillus strains which improve the health andperformance of production animals. The invention further relates to useof the Bacillus strains in animal feed and animal feed additives.

BACKGROUND OF THE INVENTION

Lawsonia intracellularis (LI) is a bacterial pathogen causing theintestine disease proliferative enteropathy (PE) in a wide range ofanimals including pigs and horses. The pathogen is spread through thefeces of the animals and causes diarrhea, depression, fever, inappetence(anorexia), weight loss, edema (fluid swelling) on the abdomen or lowerlimbs, a poor hair coat, and intermittent colic due to thickening ofmucosal lining in the small and large intestine. Furthermore, chronicforms of PE may lead to clinical or sub-clinical effects on weight gain,feed conversion and fecal consistency. Clinical observations generallyinclude diarrhea and poor Body Weight Gain of growing pigs.

Treatment of Lawsonia intracellularis infection is today limited totreatment with antibiotics. However, the treated animals do in manycases not recover fully after the treatment, and antibiotic resistanceis sometimes experienced (Jung-Yong Yeh et al., Antimicrobial Agents andChemotherapy, September 2011, p. 4451-4453).

An alternative to treatment with antibiotics is to vaccinate animalsagainst infections induced by Lawsonia intracellularis. The purpose of avaccination is to build up immunity in the animal before the firstcontact with the field pathogen. To get the best possible effect of avaccine, animals must be vaccinated as early as possible, preferably inthe first three weeks of life, to develop a reliable immunity before thefirst field infection.

There is a need for improved and sustainable methods for preventingand/or alleviating infections caused by Lawsonia intracellularis.

SUMMARY OF THE INVENTION

The invention provides Bacillus strains which have activity againstLawsonia intracellularis infection.

The invention further provides Bacillus strains having activity againstLawsonia intracellularis infection, wherein the strains reduce thenumber of heavily infected cells (HIC) in a method comprising the steps:

-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

In one aspect, body weight gain, average daily gain and/or feedconversion ratio is improved in animals which have been fed with theBacillus strains of the invention. In one alternative or further aspectof the invention, the Bacillus strains reduce the effect of inflammationon Electrical Resistance in Caco-2 cells in vitro compared to the effectof inflammation on Electrical Resistance in Caco-2 cells in vitrowithout the Bacillus strain. In a further aspect, the effect ofinflammation on Electrical Resistance is measured in a trans-epithelialelectrical resistance (TEER) test.

The Bacillus strains of the invention decrease in further aspects therelative abundance of one or more members of the phylum Proteobacteriain the intestinal microbiome of animals fed with feed comprising theBacillus strain.

In a yet further aspect, the Bacillus strains increase the relativeabundance of one or more members of a specific genera selected from thegroup consisting of: Ruminococcus, Blautia, Lactobacillus,Faecalibacterium, and Megasphera in the intestinal microbiome of animalsfed with feed comprising the Bacillus strain.

In a still further aspect of the invention, the Bacillus strains areselected from one or more Bacillus subtilis strains, one or moreBacillus licheniformis strains, one or more Bacillus pumilus strains,one or more Bacillus amyloliquefaciens strains, and any combinationthereof.

The Bacillus strains according to the invention may prevent or alleviateLawsonia intracellularis in production animals when fed to said animals.In one aspect, the production animals are selected from the groupconsisting of: pigs, swine, piglets, growing pigs, and sows.

In one aspect of the invention, the Bacillus strains of the inventionmay be in the form of spores. In a further aspect, at least 70% of theBacillus spores survive the gastric environment in a swine such as e.g.pigs, piglets, growing pigs, or sows.

The invention also provides for compositions comprising one or moreBacillus strains described herein, the use of one or more Bacillusstrains for prevention and/or alleviation of Lawsonia intracellularis inan animal, and animal feed or animal feed additives comprising one ormore Bacillus strains described herein.

Also described herein is a method for improving one or more performanceparameter(s) selected from the list consisting of body weight gain,average daily gain and feed conversion rate in an animal comprising thestep of administering one or more Bacillus strains of the invention inthe feed of the animal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of feeding 1 × 10¹² CFU probiotics (O52YJ6 or3002) / ton on average fecal scoring (score 0 = best, i.e. no diarrhea)of animals during 21 days after an oral challenge with Lawsoniaintracellularis.

FIG. 2 shows the effect of feeding piglets a diet enriched with 1 ×10^12 CFU probiotics / ton from weaning (age 21 days) to slaughter (age70 days) on average daily gain (ADG). “Challenged” indicates that allanimals in that group received an oral challenge with Lawsoniaintracelluaris (approximately 2.0 × 10^7 bacteria / dose) at age 52days.

FIG. 3 shows anti-Lawsonia intracellularis IgG in sera obtained atslaughter, i.e. 18 days after oral challenge with Lawsoniaintracellularis. All probiotics, but especially O42AH3 and O72NR7,resulted in lower IgG responses, suggesting a protective effect againstLawsonia intracellularis.

FIG. 4 shows percent animals being sero-positive for Lawsoniaintracellularis at the end of the experiment, i.e. percentage of animalshaving ELISA values > 30 at day 70 of age following an oral Lawsoniaintracellularis challenge at age 52 days.

FIG. 5 shows the effect of probiotics on fecal Lawsonia intracellularisshedding.

FIG. 6 shows macroscopic thickening in the small intestine. White partsof each bar indicate the fraction of piglets with no thickening atnecropsy, shaded parts of bars indicate the fraction of animals withthickening.

FIG. 7 shows average small intestinal microscopic scores per treatmentgroup. All three probiotics reduced infection scoring after Lawsoniaintracellularis challenge compared to non-treated animals.

FIGS. 8A and 8B show the effect of feeding weaned piglets differentdiets in a farm having presence of Lawsonia intracellularis, where theeffect is measured on average daily gain (“ADG”, in g/day) and feedconversion rate (“FCR” in g/day; daily feed intake / ADG) calculated forthe nursery phase as a whole (28 days - 70 days) or for the fatteningphase as a whole (70 days - 165 days). NC (“negative control”) refers toanimals receiving standard diet; PC to animals on standard diet but whowere all vaccinated with Enterisol within a week of weaning; O42AH3 andO72NR7 refers to groups of animals receiving standard diet to which therespective Bacillus strains had been added at an inclusion rate of 1 ×10¹² CFU / ton feed.

FIG. 9 shows anti-Lawsonia intracellularis IgG in sera obtained at day70 of piglets receiving different diets in a farm having presence ofLawsonia intracellularis. NC (“negative control”) refers to animalsreceiving standard diet; PC to animals on standard diet but who were allvaccinated with Enterisol within a week of weaning; O42AH3 and O72NR7refers to groups of animals receiving standard diet to which therespective Bacillus strains had been added at an inclusion rate of 1 ×10¹² CFU / ton feed.

FIGS. 10A and 10B show the effect of feeding weaned piglets differentdiets in a farm having presence of Lawsonia intracellularis, where theeffect is measured on average daily gain (“ADG”, in g/day) and feedconversion rate (“FCR” in g/day; daily feed intake / ADG) calculated forthe following phases 28 days - 45 days, 45 days - 88 days, 88 days - 147days and 147 days - 207 days. NC (“negative control”) refers to animalsreceiving standard diet; PC to animals on standard diet but who were allvaccinated with Enterisol within a week of weaning; O52YYT and O72NR7refers to groups of animals receiving standard diet to which therespective Bacillus strains had been added at an inclusion rate of 1 ×10¹² CFU / ton feed.

FIG. 11 shows the effect of probiotics on fecal Lawsonia intracellularisshedding at day 88 and 147 of piglets receiving different diets in afarm having presence of Lawsonia intracellularis.

FIG. 12 shows TEER results comparing DSM 32563 (O72NR7) vs. controlwithout inflammatory conditions at a Multiplicity Of Infection (MOI) of5.

FIG. 13 shows that DSM 32563 (O72NR7) rescued the inflammation-dependentdrop in TEER seen with inflammatory conditions caused by TNFalpha andINFgamma at a Multiplicity Of Infection (MOI) of 5.

DEFINITIONS

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences, and context known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in context of thedisclosure.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

Alleviate Lawsonia intracellularis infections: The term “alleviateLawsonia intracellularis infections” means a method and/or compositionthat lightens or reduces development of a Lawsonia intracellularisinfection in an animal.

Animal feed: The term “animal feed” refers to any compound, preparation,or mixture suitable for, or intended for intake by an animal. Animalfeed for a production animal comprises concentrates as well as forexample vitamins, minerals, enzymes, amino acids and/or other feedingredients (such as in a premix). The animal feed may further compriseforage.

Average Daily Gain: The Average Daily Gain (ADG) of an animal is theincrease in bodyweight of the animal per day expressed in g/day.Typically the weight of the animal is measured at a few time pointsduring the experiment and the difference in weight is then divided bythe number of days in the measured period to get the Average Daily Gain.An example of Average Daily Gain determination is given in Example 4.

Blend: the term “blend” means more than one of the bacterial strainsdescribed herein.

Body Weight Gain: The Body Weight Gain of an animal is the increase ofbody weight of the animal over a specified time period. The body weightgain can readily be determined by weighing the animal at two timepointsand calculating the gain in weight by subtracting the weight at thefirst timepoint from the weight at the second timepoint.

Composition: The term “composition” refers to a composition comprising acarrier and at least one bacterial strain as described herein. Thecompositions described herein may be mixed with an animal feed(s) toobtain a “mash feed”, extruded or pressed feed pellets, or liquid feed.

Concentrates: The term “concentrates” means feed with high protein andenergy concentrations, such as fish meal, molasses, oligosaccharides,sorghum, seeds and grains (either whole or prepared by crushing,milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed presscake (e.g., from cottonseed, safflower, sunflower, soybean (such assoybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake,yeast derived material and distillers grains (such as wet distillersgrains (WDS) and dried distillers grains with solubles (DDGS)).

Direct Fed Microbial (DFM): The term “direct fed microbial” or “DFM”means live microorganisms including spores which, when administered inadequate amounts, confer a benefit, such as improved digestion orhealth, on the host.

Effective amount/concentration/dosage: The terms “effective amount”,“effective concentration”, or “effective dosage” are defined as theamount, concentration, or dosage of the bacterial strain(s) sufficientto improve the digestion or yield of an animal. The actual effectivedosage in absolute numbers depends on factors including: the state ofhealth of the animal in question, other ingredients, additives, or drugspresent. The “effective amount”, “effective concentration”, or“effective dosage” of the bacterial strains may be determined by routineassays known to those skilled in the art.

Fed: The term “fed” means any type of oral administration such asadministration via an animal feed or via drinking water.

FCR (Feed Conversion Rate): FCR is a measure of an animal’s efficiencyin converting feed mass into increases of the desired output, where theoutput is the mass gained by the animal. The term is used for animalsthat are raised for meat, such as e.g. swine, poultry and fish.Specifically, FCR is the mass of the food eaten divided by the output,all over a specified period. Improvement in FCR means reduction of theFCR value. A FCR improvement of 2% means that the FCR was reduced by 2%.

Feeding an animal: The terms “feeding an animal” or “fed to an animal”means that the composition of the present invention is administeredorally to the animal one or more times in an effective amount. The oraladministration is typically repeated, e.g., one or more times daily overa specified time period such as several days, one week, several weeks,one months or several months. Accordingly, the terms “feeding” or “fed”mean any type of oral administration such as administration via ananimal feed or via drinking water.

Survive the gastric environment: The term “survive the gastricenvironment” is herein used for describing the ability of e.g. aBacillus spore to survive in an environment mimicking the environment inthe gastrointestinal tract in vitro. The ability to survive the gastricenvironment may be measured according to methods generally known in theart. In one embodiment, the ability to survive the gastric environmentis measured as described in T.M. Barbosa, C.R. Serra, R.M. La Ragione,M.J. Woodward, A.O. Henriques. Screening for Bacillus isolates in thebroiler gastrointestinal tract. Appl. Environ. Microbiol., 71 (2005),pp. 968-978.

IgG score: The term “IgG score” indicates to what extent immunoglobulinG directed against Lawsonia intracellularis was produced in response toan infection with Lawsonia intracellularis. A low IgG score indicates alimited infection with low morbidity, whereas a high score indicates asevere infection with higher risk of intestinal lesion formation andother morbidity.

Isolated: The term “isolated” means that the one or more bacterialstrains described herein are in a form or environment which does notoccur in nature, that is, the one or more bacterial strains are at leastpartially removed from one or more or all of the naturally occurringconstituents with which it is associated in nature.

Pellet: The terms “pellet” and/or “pelleting” refer to solid rounded,spherical and/or cylindrical tablets or pellets and the processes forforming such solid shapes, particularly feed pellets and solid extrudedanimal feed. As used herein, the terms “extrusion” or “extruding” areterms well known in the art and refer to a process of forcing acomposition, as described herein, through an orifice under pressure.

Premix: The term is used for a mixture of micro ingredients such as e.g.vitamins, minerals, enzymes, amino acids, preservatives, antibiotics,other feed ingredients or any combination thereof, and typically existsas a powder or in granulate form. The premix is mixed into the animalfeed before feeding to animals. This is opposed to e.g. macroingredients such as cereals which are usually added separately to theanimal feed.

Prevent Lawsonia intracellularis infections: The term “prevent Lawsoniaintracellularis infections” means a method and/or composition thatprevents and/or controls development of a Lawsonia intracellularisinfection in an animal.

Relative abundance: The term “relative abundance” when used inconnection with the intestinal microbiome of animals indicates theproportion of sequences matching a known and identified taxonomic groupin a sample, compared to all sequences from that sample.

Spore: The terms “spore” and “endospore” are interchangeable and havetheir normal meaning which is well known and understood by those ofskill in the art. As used herein, the term spore refers to amicroorganism in its dormant, protected state.

Stable: The term “stable” is a term that is known in the art, and in apreferred aspect, stable is intended to mean the ability of themicroorganism to remain in a spore form until it is administered to ananimal to improve the health of the animal.

Swine: The term “swine” or “pigs” means domesticated pigs kept by humansfor food, such as their meat. Swine includes members of the genus Sus,such as Sus scrofa domesticus or Sus domesticus and include piglets,growing pigs, sows, and boars.

TEER (Trans-Epithelial Electrical Resistance): The term “TEER” meansTrans-Epithelial Electrical Resistance. It is a measure of theelectrical resistance across a cell membrane and gives an indication oftight junctions or barrier function. TEER is generally used as an invitro model for the strength of the intestinal barrier function. Thegreater the electrical resistance, the stronger the barrier function.

Vegetable protein: The term “vegetable protein” refers to any compound,preparation or mixture that includes at least one protein derived fromor originating from a vegetable, including modified proteins andprotein-derivatives.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods of alleviating and/or treating ananimal against infection by Lawsonia intracellularis or similar orotherwise related microorganisms, and/or methods of preventing againstinfection by Lawsonia intracellularis or similar or otherwise relatedmicroorganisms in an animal. The method includes administering one ormore Bacillus strains to the animal in a dose sufficient to alleviate,treat and/or prevent Lawsonia intracellularis infection.

It has been surprisingly found that the addition of direct fed microbes(DFM) from Bacillus species to animal feed can be used to prevent,alleviate and/or treat Lawsonia intracellularis infections in productionanimals such as pigs and/or poultry and at the same time improve thebody weight gain (BWG), average daily gain (ADG) and/or feed conversionratio (FCR) (in both Lawsonia intracellularis challenged andunchallenged animals). Further, it was surprisingly found that Bacillusstrains may further reduce the effect of inflammation on ElectricalResistance in Caco-2 cells in vitro and/or decrease the relativeabundance of one or more members of the phylum Proteobacteria in theintestinal microbiome of animals fed with feed comprising the Bacillusstrain and/or increase the relative abundance of one or more members ofa specific genera selected from the group consisting of: Ruminococcus,Blautia, Lactobacillus, Faecalibacterium and Megasphera in theintestinal microbiome of animals fed with feed comprising the Bacillusstrain.

The inventors have identified Bacillus strains which are active againstthe pathogen Lawsonia intracellularis.

It has furthermore surprisingly been found that when using the Bacillusstrains of the invention, the ability of Lawsonia intracellularis tocause re-infection of swine is reduced.

In one embodiment, the Bacillus strains described herein decrease theshedding of Lawsonia intracellularis organisms in the feces, and/orreduce or decrease the number of gross lesions in intestinal tissue atnecropsy.

In one embodiment, the risk of developing severe diarrhea upon challengewith Lawsonia intracellularis may be indicated by determining the fecalconsistency using fecal scoring. Fecal consistency is primarily afunction of the amount of moisture in the stool and can be used toidentify changes in colonic health and other problems. Ideally, in ahealthy animal, stools should be firm but not hard, pliable andsegmented, and easy to pick up (i.e. score 0 in example 2).

The invention relates to the following aspects and embodiments withrespect to Bacillus strains:

-   Aspect 1: One or more Bacillus strains characterized in that:-   i) the Bacillus strain has activity against a Lawsonia    intracellularis infection, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or feed conversion ratio (FCR) in    animals fed with the Bacillus strain.-   Aspect 2: One or more Bacillus strains having activity against    Lawsonia intracellularis infection, wherein the strains reduce the    number of heavily infected cells (HIC) in a method comprising the    steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

In one embodiment, the Bacillus strain of the invention reduces theeffect of inflammation on Electrical Resistance in Caco-2 cells in vitrocompared to the effect of inflammation on Electrical Resistance inCaco-2 cells in vitro without the Bacillus strain. In one particularembodiment, the effect of inflammation on Electrical Resistance ismeasured in a trans-epithelial electrical resistance (TEER) test.

In one embodiment, the Bacillus strain decreases the relative abundanceof one or more members of the phylum Proteobacteria in the intestinalmicrobiome of animals fed with feed comprising the Bacillus straincompared to animals fed with the same feed without the Bacillus strain.The members of the phylum Proteobacteria are in one embodiment selectedfrom the group consisting of: Escherichia, Shigella, Campylobacter,Burkholderia, Acinetobacter and any combination thereof.

In one embodiment, the Bacillus strain increases the relative abundanceof one or more members of a specific genera selected from the groupconsisting of: Ruminococcus, Blautia, Lactobacillus, Faecalibacterium,and Megasphera in the intestinal microbiome of animals fed with feedcomprising the Bacillus strain compared to animals fed with the samefeed without the Bacillus strain.

In one embodiment the improvement in body weight gain results in a bodyweight gain of at least 0.5%, such as at least 0.8%, such as at least1.5%, such as at least 1.8%, such as at least 2.0%, such as at least2.3%, such as at least 3.5%, such as at least 4.2%, such as at least5.2%, such as at least 6.5%, such as at least 7.3%. In a preferredembodiment the improvement in body weight gain results in a body weightgain selected from the group consisting of from 1.8% to 2.0%, from 2.0%to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%,from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from7% to 10%, or any combination thereof. The body weight gain can bedetermined as explained in the definition of body weight gain.

In one embodiment the improvement in average daily gain results in anaverage daily gain of at least 0.5%, such as at least 0.8%, such as atleast 1.5%, such as at least 1.8%, such as at least 2.0%, such as atleast 2.3%, such as at least 3.5%, such as at least 4.2%, such as atleast 5.2%, such as at least 6.5%, such as at least 7.3%. In a preferredembodiment the improvement in average daily gain for results in anaverage daily gain selected from the group consisting of from 1.8% to2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6%to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from5% to 7%, from 7% to 10%, or any combination thereof. The average dailygain can be determined as described in Example 4.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 1.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 2.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 3.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 4.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 11.

In one embodiment of the invention, the Bacillus strain comprises 16SrDNA that is more than 98% (such as more than 98.5%, such as more than99%, such as more than 99.5%, such as more than 99.5%) sequence identityto SEQ ID NO: 12.

In one embodiment, the one or more Bacillus strains of the invention areselected from the group consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   e. Bacillus subtilis strain SB3175 having deposit accession number    NRRL B-50605 or a strain having all the identifying characteristics    of Bacillus subtilis strain NRRL B-50605 or a mutant thereof.

In one embodiment, the one or more Bacillus strains of the invention areselected from the group consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In one embodiment, the one or more Bacillus strains of the invention areselected from the group consisting of:

-   a. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   b. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In one embodiment, the Bacillus strain of the invention is Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof.

In one embodiment, the Bacillus strain of the invention is Bacilluspumilus strain O72NR7 having deposit accession number DSM 32563 or astrain having all the identifying characteristics of Bacillus pumilusstrain DSM 32563 or a mutant thereof.

In one embodiment, the invention relates to a composition comprisingspores of one or more Bacillus strains according to invention.

More specifically the invention relates to the following aspects andembodiments with respect to compositions comprising Bacillus strains:

-   Aspect 3: A composition comprising spores of a Bacillus strain    characterized in that:-   i) the Bacillus strain has activity against an Lawsonia    intracellularis infection, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or feed conversion ratio (FCR) in    animals fed with the Bacillus strain.-   Aspect 4: A composition comprising spores of a Bacillus strains    having activity against Lawsonia intracellularis infection, wherein    the strains reduce the number of heavily infected cells (HIC) in a    method comprising the steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

In one embodiment of the invention, the Bacillus strain of thecomposition reduces the effect of inflammation on Electrical Resistancein Caco-2 cells in vitro compared to the effect of inflammation onElectrical Resistance in Caco-2 cells in vitro without the Bacillusstrain. In one particular embodiment, the effect of inflammation onElectrical Resistance is measured in a trans-epithelial electricalresistance (TEER) test.

In one embodiment of the invention, the Bacillus strain of thecomposition decreases the relative abundance of one or more members ofthe phylum Proteobacteria in the intestinal microbiome of animals fedwith feed comprising the Bacillus strain compared to animals fed withthe same feed without the Bacillus strain. The members of the phylumProteobacteria are in one embodiment selected from the group consistingof: Escherichia, Shigella, Campylobacter, Burkholderia, Acinetobacterand any combination thereof.

In one embodiment of the invention, the Bacillus strain of thecomposition increases the relative abundance of one or more members of aspecific genera selected from the group consisting of: Ruminococcus,Blautia, Lactobacillus, Faecalibacterium, and Megasphera in theintestinal microbiome of animals fed with feed comprising the Bacillusstrain compared to animals fed with the same feed without the Bacillusstrain.

In one embodiment the improvement in body weight gain results in a bodyweight gain of at least 0.5%, such as at least 0.8%, such as at least1.5%, such as at least 1.8%, such as at least 2.0%, such as at least2.3%, such as at least 3.5%, such as at least 4.2%, such as at least5.2%, such as at least 6.5%, such as at least 7.3%. In a preferredembodiment the improvement in body weight gain results in a body weightgain selected from the group consisting of from 1.8% to 2.0%, from 2.0%to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%,from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from7% to 10%, or any combination thereof. The body weight gain can bedetermined as explained in the definition of body weight gain.

In one embodiment the improvement in food conversion rate results in afood conversion rate improvement of at least 0.5%, such as at least0.8%, such as at least 1.5%, such as at least 1.8%, such as at least2.0%, such as at least 2.3%, such as at least 3.5%, such as at least4.2%, such as at least 5.2%, such as at least 6.5%, such as at least7.3%. In a preferred embodiment, the improvement in food conversion rateresults in a food conversion rate improvement selected from the groupconsisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%,from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8%to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or anycombination thereof. The food conversion rate can be determined asexplained in the definition of food conversion rate.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 1.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 2.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 3.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 4.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 11.

In one embodiment of the invention, the Bacillus strain of thecomposition comprises 16S rDNA that is more than 98% (such as more than98.5%, such as more than 99%, such as more than 99.5%, such as more than99.5%) sequence identity to SEQ ID NO: 12.

In one embodiment of the invention the bacillus spores of thecomposition are present as dried spores such as spray-dried spores. Inone embodiment of the invention the bacillus spores of the compositionare present as stable spores. The composition according to the inventioncan also be a liquid composition and/or comprise culture supernatantcomprising one or more Bacillus strain(s) of the invention.

In one embodiment of the invention the composition further comprises acarrier. The carrier can comprise one or more of the followingcompounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or1,3-propylene glycol, sodium chloride, sodium benzoate, potassiumsorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin,glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corngluten meal, starch, cellulose farigel, cassava cores, sodium aluminiumsilicate, colloidal amorphous silica, Sipernat 50S, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000 andcarbopol.

In a preferred embodiment of the invention the composition furthercomprises calcium carbonate and sodium aluminium silicate.

In a preferred embodiment of the invention the composition furthercomprises calcium carbonate, sodium aluminium silicate and sucrose.

In another preferred embodiment of the invention the composition furthercomprises one or more carriers such as one or more carriers selectedfrom the group consisting of Calcium carbonate, sodium sulfate, starch,farigel and cassava cores.

In another preferred embodiment of the invention the composition furthercomprises one or more flowability agents such as sodium aluminiumsilicate and/or colloidal amorphous silica (e.g., Sipernat 50S).

In another preferred embodiment of the invention the composition furthercomprises one or more binder such as one or more binders selected fromthe group consisting of sucrose, sorbitol, glycerol, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000,dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacilluslicheniformis strain O42AH3 having deposit accession number DSM 32559 ora strain having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559 or a mutant thereof, calcium carbonateand sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacilluslicheniformis strain O42AH3 having deposit accession number DSM 32559 ora strain having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559 or a mutant thereof, calcium carbonate,sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacilluslicheniformis strain O42AH3 having deposit accession number DSM 32559 ora strain having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559 or a mutant thereof and one or morecarriers such as one or more carriers selected from the group consistingof calcium carbonate, sodium sulphate, starch, farigel and cassavacores.

In a preferred embodiment the composition comprises Bacilluslicheniformis strain O42AH3 having deposit accession number DSM 32559 ora strain having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559 or a mutant thereof and one or moreflowability agents such as sodium aluminium silicate and/or colloidalamorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacilluslicheniformis strain O42AH3 having deposit accession number DSM 32559 ora strain having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559 or a mutant thereof and one or morebinder such as one or more binders selected from the group consisting ofsucrose, sorbitol, glycerol, polyethylene glycol 200, polyethyleneglycol 400, polyethylene glycol 600, polyethylene glycol 1000,polyethylene glycol 1500, polyethylene glycol 4000, dextrin,maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus pumilusstrain O72NR7 having deposit accession number DSM 32563 or a strainhaving all the identifying characteristics of Bacillus pumilus strainDSM 32563 or a mutant thereof, calcium carbonate and sodium aluminiumsilicate.

In a preferred embodiment the composition comprises Bacillus pumilusstrain O72NR7 having deposit accession number DSM 32563 or a strainhaving all the identifying characteristics of Bacillus pumilus strainDSM 32563 or a mutant thereof, calcium carbonate, sodium aluminiumsilicate and sucrose.

In a preferred embodiment the composition comprises Bacillus pumilusstrain O72NR7 having deposit accession number DSM 32563 or a strainhaving all the identifying characteristics of Bacillus pumilus strainDSM 32563 or a mutant thereof and one or more carriers such as one ormore carriers selected from the group consisting of calcium carbonate,sodium sulphate, starch, farigel and cassava cores.

In a preferred embodiment the composition comprises Bacillus pumilusstrain O72NR7 having deposit accession number DSM 32563 or a strainhaving all the identifying characteristics of Bacillus pumilus strainDSM 32563 or a mutant thereof and one or more flowability agents such assodium aluminium silicate and/or colloidal amorphous silica (e.g.,Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus pumilusstrain O72NR7 having deposit accession number DSM 32563 or a strainhaving all the identifying characteristics of Bacillus pumilus strainDSM 32563 or a mutant thereof and one or more binder such as one or morebinders selected from the group consisting of sucrose, sorbitol,glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethyleneglycol 600, polyethylene glycol 1000, polyethylene glycol 1500,polyethylene glycol 4000, dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof, calciumcarbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof, calciumcarbonate, sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof and one or morecarriers such as one or more carriers selected from the group consistingof calcium carbonate, sodium sulphate, starch, farigel and cassavacores.

In a preferred embodiment the composition comprises Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof and one or moreflowability agents such as sodium aluminium silicate and/or colloidalamorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 or a strain having all the identifying characteristics of Bacillusamyloliquefaciens strain DSM 32561 or a mutant thereof and one or morebinder such as one or more binders selected from the group consisting ofsucrose, sorbitol, glycerol, polyethylene glycol 200, polyethyleneglycol 400, polyethylene glycol 600, polyethylene glycol 1000,polyethylene glycol 1500, polyethylene glycol 4000, dextrin,maltodextrin and carbopol.

In a preferred embodiment the composition according to the invention thecomposition comprises from 10⁴ to 10¹⁴ CFU/g of isolated Bacillus spores

In a further embodiment, the composition according to the inventioncomprises one or more bacterial strains such as at least two of theabove strains up to and including all of the strains in the groupconsisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   e. Bacillus subtilis strain SB3175 having deposit accession number    NRRL B-50605 or a strain having all the identifying characteristics    of Bacillus subtilis strain NRRL B-50605 or a mutant thereof.

In a further embodiment, the composition according to the inventioncomprises one or more bacterial strains such as at least two of theabove strains up to and including all of the strains in the groupconsisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In a further embodiment, the composition according to the inventioncomprises one or more bacterial strains such as at least two of theabove strains up to and including all of the strains in the groupconsisting of:

-   a. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   b. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In an embodiment to any of the aforementioned embodiments, developmentof severe diarrhea is prevented after 24 hours, such as after 36 hours,2 days, 3 days, 4 days, 5 days, 6 days or 1 week of feeding the Bacillusspore to the animal.

In another embodiment to any of the aforementioned embodiments,development of severe diarrhea is prevented after 2 weeks, such as after3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months or 1 year of feeding the Bacillus spore to the animal.

In an embodiment to any of the aforementioned embodiments, shedding ofLawsonia intracellularis in feces is decreased for animals fed with theBacillus spore compared to animals not fed with Bacillus strains.

In a further embodiment, shedding of Lawsonia intracellularis in fecesis decreased after feeding the Bacillus spore to the animal for at least24 hours, such as at least 36 hours, 2 days, 3 days, 4 days, 5 days, 6days or 1 week.

In another embodiment to any of the aforementioned embodiments, sheddingof Lawsonia intracellularis in feces is decreased after feeding theBacillus spore to the animal for at least 2 weeks, such as at least 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months or 1 year.

In an embodiment to any of the aforementioned embodiments, lesions inthe intestinal tract of the animal are reduced after feeding theBacillus strain to the animal for at least 24 hours, such as at least 36hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week compared tolesions in the intestinal tract of an animal not fed with Bacillusstrains.

In another embodiment to any of the aforementioned embodiments, lesionsin the intestinal tract of the animal are reduced after feeding theBacillus strain to the animal for at least 2 weeks, such as at least 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months or 1 year compared to lesions in the intestinal tract of ananimal not fed with Bacillus strains.

In an embodiment to any of the aforementioned embodiments, the IgG scorein the animal is reduced after feeding the Bacillus strain to the animalfor at least 24 hours, such as at least 36 hours, 2 days, 3 days, 4days, 5 days, 6 days or 1 week compared to the IgG score in animals notfed with Bacillus strains.

In another embodiment to any of the aforementioned embodiments, the IgGscore in the animal is reduced after feeding the Bacillus strain to theanimal for at least 2 weeks, such as at least 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, 6 months or 1 year compared tothe IgG score in animals not fed with Bacillus strains.

In an embodiment to any of the aforementioned embodiments, Average DailyGain of the animal is increased after feeding the Bacillus strain to theanimal for at least 24 hours, such as at least 36 hours, 2 days, 3 days,4 days, 5 days, 6 days or 1 week compared to the Average Daily Gain ofanimals not fed with Bacillus strains.

In another embodiment to any of the aforementioned embodiments, AverageDaily Gain of the animal is increased after feeding the Bacillus strainto the animal for at least 2 weeks, such as at least 3 weeks, 4 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 yearcompared to the Average Daily Gain of animals not fed with Bacillusstrains.

In an embodiment to any of the aforementioned embodiments, FeedConversion Rate (FCR) of the animal is improved after feeding theBacillus strain to the animal for at least 24 hours, such as at least 36hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week compared to theAverage Daily Gain of animals not fed with Bacillus strains.

In another embodiment to any of the aforementioned embodiments, FeedConversion Rate (FCR) of the animal is improved after feeding theBacillus strain to the animal for at least 2 weeks, such as at least 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months or 1 year compared to the Average Daily Gain of animals not fedwith Bacillus strains.

In an embodiment to any of the aforementioned embodiments, the risk ofdeveloping Proliferative Hemorrhagic Enteropathy (PHE) is reduced after24 hours, such as after 36 hours, 2 days, 3 days, 4 days, 5 days, 6 daysor 1 week of feeding the Bacillus spore to the animal.

In another embodiment to any of the aforementioned embodiments, the riskof developing Proliferative Hemorrhagic Enteropathy (PHE) is reducedafter 2 weeks, such as after 3 weeks, 4 weeks, 1 month, 2 months, 3months, 4 months, 5 months, 6 months or 1 year of feeding the Bacillusspore to the animal.

In another embodiment of the invention the composition further comprisesone or more additional microbes. In another embodiment of the inventionthe composition further comprises one or more additional vaccines suchas e.g. Enterisol. In another embodiment of the invention thecomposition further comprises one or more enzymes. In another embodimentof the invention the composition further comprises one or more vitamins.In another embodiment of the invention the composition further comprisesone or more minerals. In another embodiment of the invention thecomposition further comprises one or more amino acids. In anotherembodiment of the invention the composition further comprises one ormore other feed ingredients.

In an embodiment to any of the aforementioned embodiments, thecomposition also improves the health of the production animal when fedto said animal. In an embodiment to any of the aforementionedembodiments, the composition increases the meat yield of the productionanimal when fed to said animal.

In a preferred embodiment, the composition comprises one or morebacterial strains described herein, wherein the bacterial count of eachof the bacterial strains is between 1×10⁴ and 1×10¹² CFU/kg ofcomposition, preferably between 1×10⁷ and 1×10¹¹ CFU/kg of composition,more preferably between 1×10⁸ and 1×10¹⁰ CFU/kg of composition and mostpreferably between 1×10⁹ and 1×10¹⁰ CFU/kg of composition.

In a preferred embodiment, the bacterial count of each of the bacterialstrains in the composition is between 1×10⁴ and 1×10¹² CFU/kg of drymatter, preferably between 1×10⁷ and 1×10¹¹ CFU/kg of dry matter, morepreferably between 1×10⁸and 1×10¹¹ CFU/kg of dry matter and mostpreferably between 1×10⁸ and 1×10¹⁰ CFU/kg of dry matter. In a morepreferred embodiment the bacterial count of each of the bacterialstrains in the composition is between 1×10⁹ and 1×10¹⁰ CFU/kg of drymatter

In a preferred embodiment, the composition has a bacterial count of eachBacillus spore between 1×10³ and 1×10¹³ CFU/animal/day, preferablybetween 1×10⁵ and 1×10¹¹ CFU/animal/day, more preferably between 1×10⁶and 1×10¹⁰ CFU/animal/day and most preferably between 1×10⁷ and 1×10⁹CFU/animal/day.

In still yet another embodiment of the invention, the one or morebacterial strains are present in the composition in form of a spore suchas a stable spore. In still a further embodiment of the invention, thestable spore will germinate in the intestine and/or stomach of themono-gastric animal.

In one embodiment, the one or more bacterial strains are stable whensubjected to pressures applied/achieved during an extrusion process forpelleting. In a particular embodiment, the one or more bacterial strainsare stable at pressures ranging from 1 bar to 40 bar, particularly 10bar to 40 bar, more particularly 15 bar to 40 bar, even moreparticularly 20 bar to 40 bar, still even more particularly 35 bar to 37bar, even still more particularly 36 bar.

In a particular embodiment, the one or more bacterial strains are stableat high temperatures. In particular, the bacterial strains are stablewhen they are subjected to temperatures achieved during an extrusionprocess for pelleting. In an even more particular embodiment, the one ormore bacterial strains are stable at temperatures ranging from 60° C. to120° C., particularly temperatures ranging from, 90° C. to 120° C., evenmore particularly temperatures ranging from 95° C. to 120° C.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of a strain selected from the groupconsisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   e. Bacillus subtilis strain SB3175 having deposit accession number    NRRL B-50605 or a strain having all the identifying characteristics    of Bacillus subtilis strain NRRL B-50605 or a mutant thereof.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of a strain selected from the groupconsisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of a strain selected from the groupconsisting of:

-   a. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   b. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of Bacillus amyloliquefaciens strainO52YYT having deposit accession number DSM 32561 or a strain having allthe identifying characteristics of Bacillus amyloliquefaciens strain DSM32561 or a mutant thereof.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of Bacillus pumilus strain O72NR7 havingdeposit accession number DSM 32563 or a strain having all theidentifying characteristics of Bacillus pumilus strain DSM 32563 or amutant thereof.

In an embodiment, the composition further comprises one or moreadditional microbes. In a particular embodiment, the composition furthercomprises a bacterium from one or more of the following genera:Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus,Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium,Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

In a particular embodiment, the composition further comprises abacterium from one or more of the following strains of Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacilluspolymyxa, Bacillus licheniformis, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, or any combination thereof.

In a particular embodiment, the composition further comprises one ormore types of yeast. The one or more types of yeast can be selected fromthe group consisting of Saccharomycetaceae, Saccharomyces (such as S.cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus andK. lactis), Candida (such as C. utilis, also called Torula yeast),Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii),Phaffia yeasts and Basidiomycota.

In an embodiment to any of the aforementioned embodiments thecomposition further comprises a carrier. The carrier can comprise one ormore of the following compounds: water, glycerol, ethylene glycol,1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodiumbenzoate, potassium sorbate, sodium sulfate, potassium sulfate,magnesium sulfate, sodium thiosulfate, calcium carbonate, sodiumcitrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose,wheat flour, wheat bran, corn gluten meal, starch,cellulose, farigel,cassava cores, sodium aluminium silicate, colloidal amorphous silica,Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400,polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol1500, polyethylene glycol 4000 and carbopol.

In another embodiment, the composition described herein can optionallyinclude one or more enzymes. Enzymes can be classified on the basis ofthe handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also theENZYME site at the internet: www.expasy.ch/enzyme/. ENZYME is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB),Academic Press, Inc., 1992, and it describes each type of characterizedenzyme for which an EC (Enzyme Commission) number has been provided(Bairoch, The ENZYME database, 2000, Nucleic Acids Res. 28:304-305).This IUB-MB Enzyme nomenclature is based on their substrate specificityand occasionally on their molecular mechanism; such a classificationdoes not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase andalpha-galactosidase, in families based on amino acid sequencesimilarities has been proposed a few years ago. They currently fall into90 different families: See the CAZy(ModO) internet site (Coutinho, P.M.& Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL:http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html (corresponding papers:Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-active enzymes: anintegrated database approach. In “Recent Advances in CarbohydrateBioengineering”, H.J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., The Royal Society of Chemistry, Cambridge, pp. 3-12; Coutinho,P.M. & Henrissat, B. (1999) The modular structure of cellulases andother carbohydrate-active enzymes: an integrated database approach. In“Genetics, Biochemistry and Ecology of Cellulose Degradation”., K.Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimuraeds., Uni Publishers Co., Tokyo, pp. 15-23).

Thus the composition of the invention may also comprise at least oneother enzyme selected from the group comprising of phytase (EC 3.1.3.8or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1(EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixturethereof.

In a particular embodiment, the composition of the invention comprises aphytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially availablephytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™(DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue(AB Enzymes), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont).Other preferred phytases include those described in, e.g., WO 98/28408,WO 00/43503, and WO 03/066847.

In a particular embodiment, the composition of the invention comprises axylanase (EC 3.2.1.8). Examples of commercially available xylanasesinclude Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT andBarley (AB Vista), Xylathin® (Verenium), Axtra® XB(Xylanase/beta-glucanase, DuPont), Rovabio® excel (Adisseo), and RovabioAdvance® (Adisseo).

In a particular embodiment, the composition of the invention comprises aprotease (EC 3.4). Examples of commercially available proteases includeRonozyme® ProAct (DSM Nutritional Products).

The composition of the invention can, e.g., be manufactured as mashcomposition (non-pelleted) or pelleted composition. The bacteriacultures, such a e.g. Bacillus strains, and optionally enzymes can beadded as solid or liquid formulations. For example, for mash compositiona solid or liquid culture formulation may be added before or during theingredient mixing step. Typically, a liquid culture preparationcomprises the culture of the invention optionally with a polyol, such asglycerol, ethylene glycol or propylene glycol, and is added after thepelleting step, such as by spraying the liquid formulation onto thepellets.

The enzyme may be added to the composition as a granule, which isoptionally pelleted or extruded. The granule typically comprises a coreparticle and one or more coatings, which typically are salt and/or waxcoatings. The core particle can either be a homogeneous blend of anactive compound optionally together with salts (e.g., organic orinorganic zinc or calcium salt) or an inert particle with an activecompound applied onto it. The active compound is the culture of theinvention optionally combined with one or more enzymes. The inertparticle may be water soluble or water insoluble, e.g., starch, a sugar(such as sucrose or lactose), or a salt (such as NaCl, Na₂SO₄). The saltcoating is typically at least 1 µm thick and can either be oneparticular salt or a mixture of salts, such as Na₂SO₄, K₂SO₄, MgSO₄and/or sodium citrate. Other examples are those described in, e.g., WO2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO00/70034 or polymer coating such as described in WO 01/00042.

The present invention also relates to animal feed additives comprisingone or more Bacillus strains. Thus, in one embodiment, the inventionrelates to an animal feed additive comprising a Bacillus strain,wherein:

-   i) the Bacillus strain has activity against an Lawsonia    intracellularis infection, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or feed conversion ratio (FCR) in    animals fed with the Bacillus strain.

In another aspect, the invention relates to animal feed additivescomprising one or more Bacillus strains, which is selected from thegroup consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   e. Bacillus subtilis strain SB3175 having deposit accession number    NRRL B-50605 or a strain having all the identifying characteristics    of Bacillus subtilis strain NRRL B-50605 or a mutant thereof.

In another aspect, the invention relates to animal feed additivescomprising one or more Bacillus strains, which is selected from thegroup consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32563 or a mutant thereof.

In another aspect, the invention relates to animal feed additivescomprising one or more Bacillus strains, which is selected from thegroup consisting of:

-   a. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   b. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In another aspect, the invention relates to animal feed additivescomprising Bacillus amyloliquefaciens strain O52YYT having depositaccession number DSM 32561 or a strain having all the identifyingcharacteristics of Bacillus amyloliquefaciens strain DSM 32561 or amutant thereof.

In another aspect, the invention relates to animal feed additivescomprising Bacillus pumilus strain O72NR7 having deposit accessionnumber DSM 32563 or a strain having all the identifying characteristicsof Bacillus pumilus strain DSM 32563 or a mutant thereof.

In an embodiment, the amount of Bacillus strain in the animal feedadditive is between 0.001% and 10% by weight of the composition.

In an embodiment, the animal feed additive comprises one or moreformulating agents, preferably as described herein above.

In an embodiment, the animal feed additive comprises one or more furtherenzymes, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreadditional probiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morevitamins, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreminerals, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more aminoacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or moreprebiotics, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or more organicacids, preferably as described herein below.

In an embodiment, the animal feed additive comprises one or morephytogenics, preferably as described herein below.

The present invention also relates to animal feed compositionscomprising Bacillus strains of the invention. In one embodiment, theinvention relates to an animal feed comprising the granule as describedherein and plant based material. In one embodiment, the inventionrelates to an animal feed comprising the animal feed additive asdescribed herein and plant based material.

Animal feed compositions or diets have a relatively high content ofprotein. Swine diets can be characterized as indicated in Table B of WO01/58275, columns 2-3.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneBacillus strain as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolizable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolizable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg) = N (g/kg) x 6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington DC).

Metabolizable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Distillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

The animal feed may comprise vegetable proteins. In particularembodiments, the protein content of the vegetable proteins is at least10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may bederived from vegetable protein sources, such as legumes and cereals, forexample, materials from plants of the families Fabaceae (Leguminosae),Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupinmeal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean. In another particular embodiment, the vegetable proteinsource is material from one or more plants of the family Chenopodiaceae,e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetableprotein sources are rapeseed, and cabbage. In another particularembodiment, soybean is a preferred vegetable protein source. Otherexamples of vegetable protein sources are cereals such as barley, wheat,rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Bacillusstrains can be added as solid or liquid Bacillus formulations. Forexample, for mash feed a solid or liquid Bacillus formulation may beadded before or during the ingredient mixing step. For pelleted feed the(liquid or solid) Bacillus preparation may also be added before orduring the feed ingredient step. Typically, a liquid Bacilluspreparation comprises the Bacillus strain of the invention optionallywith a polyol, such as glycerol, ethylene glycol or propylene glycol,and is added after the pelleting step, such as by spraying the liquidformulation onto the pellets. The Bacillus strain may also beincorporated in a feed additive or premix.

Alternatively, the Bacillus strain can be prepared by freezing a mixtureof Bacillus solution with a bulking agent such as ground soybean meal,and then lyophilizing the mixture.

The final Bacillus strain concentration in the diet is within the rangeof 0.01-200 mg Bacillus strain per kg diet, preferably between 0.05-100mg/kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mgBacillus strain per kg animal diet.

It is at present contemplated that the Bacillus strain is administeredin one or more of the following amounts (dosage ranges): 0.01-200;0.05-100; 0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; or 1-10; - all theseranges being in mg Bacillus strains per kg feed (ppm).

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

Thus, in a further aspect, the present invention also relates to ananimal feed comprising one or more Bacillus strains and plant basedmaterial. In another aspect, the present invention also relates to ananimal feed comprising the animal feed additive of the invention (asdescribed herein above) and plant based material.

In one embodiment, the invention relates to an animal feed comprisingplant based material and one or more Bacillus strains such as two ormore up to and including all of the strains in the group consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   e. Bacillus subtilis strain SB3175 having deposit accession number    NRRL B-50605 or a strain having all the identifying characteristics    of Bacillus subtilis strain NRRL B-50605 or a mutant thereof.

In one embodiment, the invention relates to an animal feed comprisingplant based material and one or more Bacillus strains such as two ormore up to and including all of the strains in the group consisting of:

-   a. Bacillus licheniformis strain O42AH3 having deposit accession    number DSM 32559 or a strain having all the identifying    characteristics of Bacillus licheniformis strain DSM 32559 or a    mutant thereof,-   b. Bacillus subtilis strain O52YJ6 having deposit accession number    DSM 32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   c. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   d. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In one embodiment, the invention relates to an animal feed comprisingplant based material and one or more Bacillus strains, such as twoBacillus strains, selected from the group consisting of:

-   a. Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   b. Bacillus pumilus strain O72NR7 having deposit accession number    DSM 32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.

In one embodiment, the invention relates to an animal feed comprisingplant based material and Bacillus amyloliquefaciens strain O52YYT havingdeposit accession number DSM 32561 or a strain having all theidentifying characteristics of Bacillus amyloliquefaciens strain DSM32561 or a mutant thereof.

In one embodiment, the invention relates to an animal feed comprisingplant based material and Bacillus pumilus strain O72NR7 having depositaccession number DSM 32563 or a strain having all the identifyingcharacteristics of Bacillus pumilus strain DSM 32563 or a mutantthereof.

In a preferred embodiment, the animal feed comprises one or morebacterial strains described herein, wherein the bacterial count of eachof the bacterial strains is between 1×10⁴ and 1×10¹² CFU/kg of animalfeed, preferably between 1×10⁷ and 1×10¹¹ CFU/kg of animal feed, morepreferably between 1×10⁸ and 1×10¹⁰ CFU/kg of animal feed and mostpreferably between 1×10⁸and 1×10⁹ CFU/kg of animal feed.

In a preferred embodiment, the bacterial count of each of the bacterialstrains in the animal feed is between 1×10⁴ and 1×10¹² CFU/kg of drymatter, preferably between 1×10⁶ and 1×10¹¹ CFU/kg of dry matter, morepreferably between 1×10⁸and 1×10¹⁰ CFU/kg of dry matter and mostpreferably between 1×10⁸ and 1×10⁹ CFU/kg of dry matter.

In a preferred embodiment, the animal feed has a bacterial count of eachBacillus spore between 1×10³ and 1×10¹³ CFU/animal/day, preferablybetween 1×10⁵ and 1×10¹¹ CFU/animal/day, more preferably between 1×10⁶and 1×10¹⁰ CFU/animal/day and most preferably between 1×10⁷ and 1×10⁹CFU/animal/day.

In an embodiment, the plant based material is selected from the groupconsisting of legumes, cereals, oats, rye, barley, wheat, maize, corn,sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean,wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjimbean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil,peanut, Spanish peanut, canola, rapeseed (oilseed rape), rice, beet,cabbage, sugar beet, spinach, quinoa, or pea, in a processed formthereof (such as soybean meal, rapeseed meal) or any combinationthereof.

In a further embodiment, the animal feed has been pelleted.

Preferred embodiments of the invention are described in the set of itemsherein below

-   1. One or more Bacillus strains characterized in that:-   i) the Bacillus strain has activity against Lawsonia intracellularis    infection, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or Feed Conversion Ratio (FCR) in    animals fed with the Bacillus strain.-   2. One or more Bacillus strains having activity against Lawsonia    intracellularis infection, wherein the strains reduce the number of    heavily infected cells (HIC) in a method comprising the steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains; wherein the number of HIC is    reduced at least 30% compared to the control cells.-   3. The Bacillus strain according to item 1 or 2, wherein the    Bacillus strain reduces the effect of inflammation on Electrical    Resistance in Caco-2 cells in vitro compared to the effect of    inflammation on Electrical Resistance in Caco-2 cells in vitro    without the Bacillus strain, wherein the effect of inflammation on    Electrical Resistance is measured in a trans-epithelial electrical    resistance (TEER) test.-   4. The Bacillus strain according to any one of items 1 to 3, wherein    the Bacillus strain decreases the relative abundance of one or more    members of the phylum Proteobacteria in the intestinal microbiome of    animals fed with feed comprising the Bacillus strain compared to    animals fed with the same feed without the Bacillus strain.-   5. The Bacillus strain according to item 4 wherein the one or more    members of the phylum Proteobacteria is selected from the group    consisting of: Escherichia, Shigella, Campylobacter, Burkholderia,    Acinetobacter and any combination thereof.-   6. The Bacillus strain according to any one of the preceding items,    wherein the Bacillus strain increases the relative abundance of one    or more members of a specific genera selected from the group    consisting of: Ruminococcus, Blautia, Lactobacillus,    Faecalibacterium, and Megasphera in the intestinal microbiome of    animals fed with feed comprising the Bacillus strain compared to    animals fed with the same feed without the Bacillus strain.-   7. The Bacillus strain according to any one of the preceding items,    wherein lesions in the intestinal tract of an animal are reduced    after feeding the Bacillus strain to the animal for at least 24    hours, such as at least 36 hours, 2 days, 3 days, 4 days, 5 days, 6    days or 1 week, where the lesions are reduced compared to lesions in    the intestinal tract of an animal not fed with Bacillus strains.-   8. The Bacillus strain according to any one of the preceding items,    wherein lesions in the intestinal tract of an animal are reduced    after feeding the Bacillus strain to the animal for at least 2    weeks, such as at least 3 weeks, 4 weeks, 1 month, 2 months, 3    months, 4 months, 5 months, 6 months or 1 year, where the lesions    are reduced compared to lesions in the intestinal tract of an animal    not fed with Bacillus strains.-   9. The Bacillus strain according to any one of the preceding items,    wherein the Bacillus strain is selected from Bacillus pumilus O72NR7    having deposit accession number DSM 32563, Bacillus licheniformis    O42AH3 having deposit accession number DSM 32559 and any combination    thereof.-   10. The Bacillus strain according to any one of the preceding items,    wherein the Bacillus strain comprises 16S rDNA that is more than 98%    sequence identity to SEQ ID NO: 1 and/or more than 98% sequence    identity to SEQ ID NO: 2 and/or more than 98% sequence identity to    SEQ ID NO: 3 and/or more than 98% sequence identity to SEQ ID NO: 4    and/or more than 98% sequence identity to SEQ ID NO: 11 and/or more    than 98% sequence identity to SEQ ID NO: 12.-   11. The Bacillus strains according to any one of the preceding items    to the extent possible, wherein the Bacillus strain(s) is one or    more Bacillus subtilis strains, one or more Bacillus licheniformis    strains, one or more Bacillus pumilus strains or one or more    Bacillus amyloliquefaciens strains, and any combination thereof.-   12. The Bacillus strains according to any one of the preceding items    to the extent possible, wherein the Bacillus strain(s) is one or    more Bacillus subtilis strains, one or more Bacillus licheniformis    strains and/or one or more Bacillus pumilus strains.-   13. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is one or more Bacillus subtilis    strains.-   14. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is one or more Bacillus    licheniformis strains.-   15. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is one or more Bacillus pumilus    strains.-   16. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is selected from the group    consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   17. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is selected from the group    consisting of:-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.-   18. One or more isolated Bacillus strains selected from the group    consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   19. One or more isolated Bacillus strains selected from the group    consisting of:-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof, and-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof.-   20. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is non-hemolytic.-   21. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain has activity against Lawsonia    intracellularis as determined in Example 4.-   22. The Bacillus strains according to any one of the preceding    items, wherein Lawsonia intracellularis is prevented or alleviated    in production animals.-   23. The Bacillus strains according to any one of the preceding    items, wherein the production animals are selected from the group    consisting of: Horses, guinea pigs, swine, pigs, piglets, growing    pigs, sows, boars, hamsters, and monogastric animals.-   24. The Bacillus strains according to any one of the preceding    items, wherein the production animals are selected from the group    consisting of: pigs, swine, piglets, growing pigs, and sows.-   25. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is the Bacillus licheniformis    strain O42AH3 having deposit accession number DSM 32559 or a strain    having all the identifying characteristics of Bacillus licheniformis    strain DSM 32559 or a mutant thereof.-   26. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is the Bacillus subtilis strain    O52YJ6 having deposit accession number DSM 32560 or a strain having    all the identifying characteristics of Bacillus subtilis strain DSM    32560 or a mutant thereof.-   27. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is the Bacillus amyloliquefaciens    strain O52YYT having deposit accession number DSM 32561 or a strain    having all the identifying characteristics of Bacillus    amyloliquefaciens strain DSM 32561 or a mutant thereof.-   28. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus strain is the Bacillus pumilus strain    O72NR7 having deposit accession number DSM 32563 or a strain having    all the identifying characteristics of Bacillus pumilus strain DSM    32563 or a mutant thereof.-   29. The Bacillus strains according to any one of the preceding    items, wherein the Bacillus spores of the Bacillus strains are    present as dried spores.-   30. The Bacillus strains according to any one of the preceding    items, wherein at least 70% (such as at least 80% or at least 90%)    of the Bacillus spores survive the gastric environment in a swine    such as e.g. pigs, piglets, growing pigs, or sows.-   31. The Bacillus strains according to any one of the preceding items    which are derived from a substantially pure culture.-   32. A Bacillus strain which is the Bacillus licheniformis strain    O42AH3 having deposit accession number DSM 32559 or a strain having    all the identifying characteristics of Bacillus licheniformis strain    DSM 32559 or a mutant thereof.-   33. A Bacillus strain which is the Bacillus subtilis strain O52YJ6    having deposit accession number DSM 32560 or a strain having all the    identifying characteristics of Bacillus subtilis strain DSM 32560 or    a mutant thereof.-   34. A Bacillus strain which is the Bacillus amyloliquefaciens strain    O52YYT having deposit accession number DSM 32561 or a strain having    all the identifying characteristics of Bacillus amyloliquefaciens    strain DSM 32561 or a mutant thereof.-   35. A Bacillus strain which is the Bacillus pumilus strain O72NR7    having deposit accession number DSM 32563 or a strain having all the    identifying characteristics of Bacillus pumilus strain DSM 32563 or    a mutant thereof.-   36. A composition comprising one or more Bacillus strains, wherein    the Bacillus strain is characterized in that:-   i) the Bacillus strain has activity against Lawsonia intracellularis    infection, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or Feed Conversion Ratio (FCR) in    animals fed with the Bacillus strain.-   37. A composition comprising one or more Bacillus strains having    activity against Lawsonia intracellularis infection, wherein the    strains of the composition reduce the number of heavily infected    cells (HIC) in a method comprising the steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

-   38. The composition according to item 36 or 37, wherein the Bacillus    strain of the composition reduces the effect of inflammation on    Electrical Resistance in Caco-2 cells in vitro compared to the    effect of inflammation on Electrical Resistance in Caco-2 cells in    vitro without the Bacillus strain, wherein the effect of    inflammation on Electrical Resistance is measured in a    trans-epithelial electrical resistance (TEER) test.-   39. The composition according to any one of items 36 to 38, wherein    the Bacillus strain of the composition decreases the relative    abundance of one or more members of the phylum Proteobacteria in the    intestinal microbiome of animals fed with a composition comprising    the Bacillus strain compared to animals fed with the same    composition without the Bacillus strain.-   40. The composition according to item 39, wherein the one or more    members of the phylum Proteobacteria is selected from the group    consisting of: Escherichia, Shigella, Campylobacter, Burkholderia,    Acinetobacter and any combination thereof.-   41. The composition according to any one of items 36 to 40, wherein    the Bacillus strain of the composition increases the relative    abundance of one or more members of a specific genera selected from    the group consisting of: Ruminococcus, Blautia, Lactobacillus,    Faecalibacterium, and Megasphera in the intestinal microbiome of    animals fed with a composition comprising the Bacillus strain    compared to animals fed with the same a composition without the    Bacillus strain.-   42. The composition according to any one of items 36 to 41, wherein    lesions in the intestinal tract of an animal are reduced after    feeding the composition to the animal for at least 24 hours, such as    at least 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week,    where the lesions are reduced compared to lesions in the intestinal    tract of an animal not fed with a composition comprising a Bacillus    strain.-   43. The composition according to any one of items 36 to 42, wherein    lesions in the intestinal tract of an animal are reduced after    feeding the composition to the animal for at least 2 weeks, such as    at least 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5    months, 6 months or 1 year, where the lesions are reduced compared    to lesions in the intestinal tract of an animal not fed with a    composition comprising a Bacillus strain.-   44. The composition according to any one of items 36 to 43, wherein    the one or more Bacillus strains is selected from the group    consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   45. A composition comprising one or more Bacillus strains selected    from the group consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   46. The composition according to any one of items 36 to 45, wherein    the one or more Bacillus strains are in spore form.-   47. The composition according to any one of items 36 to 46, which    further comprises calcium carbonate.-   48. The composition according to any one of items 36 to 47, wherein    the Bacillus strains are derived from a substantially pure culture.-   49. Use of one or more Bacillus strains for prevention and/or    alleviation of Lawsonia intracellularis in an animal.-   50. The use of one or more Bacillus strains according to item 49,    wherein the Bacillus strains have activity against Lawsonia    intracellularis infection and reduce the number of heavily infected    cells (HIC) in a method comprising the steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

-   51. The use of one or more Bacillus strains according to item 49 or    50, wherein-   i) the Bacillus strain has activity against Lawsonia    intracellularis, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or feed conversion ratio (FCR) in    animals fed with the Bacillus strain.-   52. The use of one or more Bacillus strains according to any one of    items 49 to 51, wherein the Bacillus strain reduces the effect of    inflammation on Electrical Resistance in Caco-2 cells in vitro    compared to the effect of inflammation on Electrical Resistance in    Caco-2 cells in vitro without the Bacillus strain, wherein the    effect of inflammation on Electrical Resistance is measured in a    trans-epithelial electrical resistance (TEER) test.-   53. The use of one or more Bacillus strains according to any one of    items 49 to 52, wherein the Bacillus strain decreases the relative    abundance of one or more members of the phylum Proteobacteria in the    intestinal microbiome of animals fed with feed comprising the    Bacillus strain compared to animals fed with the same feed without    the Bacillus strain.-   54. The use of one or more Bacillus strains according to item 53,    wherein the one or more members of the phylum Proteobacteria is    selected from the group consisting of: Escherichia, Shigella,    Campylobacter, Burkholderia, Acinetobacter and any combination    thereof.-   55. The use of one or more Bacillus strains according to any one of    items 49 to 54, wherein the Bacillus strain increases the relative    abundance of one or more members of a specific genera selected from    the group consisting of: Ruminococcus, Blautia, Lactobacillus,    Faecalibacterium, and Megasphera in the intestinal microbiome of    animals fed with feed comprising the Bacillus strain compared to    animals fed with the same feed without the Bacillus strain.-   56. The use of one or more Bacillus strains according to any one of    items 49 to 55, wherein lesions in the intestinal tract of an animal    are reduced after feeding the Bacillus strain to the animal for at    least 24 hours, such as at least 36 hours, 2 days, 3 days, 4 days, 5    days, 6 days or 1 week, where the lesions are reduced compared to    lesions in the intestinal tract of an animal not fed with Bacillus    strains.-   57. The use of one or more Bacillus strains according to any one of    items 49 to 56, wherein lesions in the intestinal tract of an animal    are reduced after feeding the Bacillus strain to the animal for at    least 2 weeks, such as at least 3 weeks, 4 weeks, 1 month, 2 months,    3 months, 4 months, 5 months, 6 months or 1 year, where the lesions    are reduced compared to lesions in the intestinal tract of an animal    not fed with Bacillus strains.-   58. The use of one or more Bacillus strains according to any one of    items 49 to 57, wherein the one or more Bacillus strains is selected    from the group consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   59. The use of one or more Bacillus strains according to any one of    items 49 to 58, wherein the Bacillus strain is non-hemolytic.-   60. Use of one or more Bacillus strains for decreasing the relative    abundance of one or more members of the phylum Proteobacteria in the    intestinal microbiome of a production animal fed with feed    comprising the Bacillus strain compared to a production animal fed    with the same feed without the Bacillus strain and/or increasing the    relative abundance of one or more members of a specific genera    selected from the group consisting of: Ruminococcus, Blautia,    Lactobacillus, Faecalibacterium, and Megasphera in the intestinal    microbiome of a production animal fed with feed comprising the    Bacillus strain compared to a production animal fed with the same    feed without the Bacillus strain, wherein the production animal is a    ruminant or a non-ruminant such as sheep, goat, cattle, cow, young    calve, deer, yank, camel, llama, kangaroo, pig, swine, turkey, duck,    chicken, horse, fish or crustacean.-   61. An animal feed or feed additive comprising one or more Bacillus    strains, wherein the Bacillus strain is characterized in that:-   i) the Bacillus strain has activity against Lawsonia    intracellularis, and-   ii) the Bacillus strain improves body weight gain (BWG) and/or    average daily gain (ADG) and/or feed conversion ratio (FCR) in    animals fed with the Bacillus strain.-   62. An animal feed or the animal feed additive comprising one or    more Bacillus strains having activity against Lawsonia    intracellularis infection, wherein the strains reduce the number of    heavily infected cells (HIC) in a method comprising the steps:-   i) preparing a filtered cell-free extract comprising the Bacillus    strain(s), Tryptic soy broth with yeast extract (TSBYE) and heat    killed E. coli;-   ii) from the extract of step i) preparing Bacillus supernatant    dilutions comprising Lawsonia intracellularis;-   iii) incubating the dilutions of step ii);-   iv) adding the incubated dilutions of step iii) to murine fibroblast    McCoy host cells;-   v) incubating the host cells of step iv) to allow for Lawsonia    intracellularis to infect the McCoy host cells;-   vi) counting the number of heavily infected cells (HIC); and-   vii) comparing the count from step vi) to the count of HIC in    control cells prepared according to steps i) to v) but not    comprising the Bacillus strains;

wherein the number of HIC is reduced at least 30% compared to thecontrol cells.

-   63. The animal feed or the animal feed additive according to item 61    or 62, wherein the Bacillus strain of the composition reduces the    effect of inflammation on Electrical Resistance in Caco-2 cells in    vitro compared to the effect of inflammation on Electrical    Resistance in Caco-2 cells in vitro without the Bacillus strain,    wherein the effect of inflammation on Electrical Resistance is    measured in a trans-epithelial electrical resistance (TEER) test.-   64. The animal feed or the animal feed additive according to any one    of items 61 to 63, wherein the Bacillus strain of the animal feed or    the animal feed additive decreases the relative abundance of one or    more members of the phylum Proteobacteria in the intestinal    microbiome of animals fed with feed comprising the Bacillus strain    compared to animals fed with the same feed without the Bacillus    strain.-   65. The animal feed or the animal feed additive according to item    64, wherein the one or more members of the phylum Proteobacteria is    selected from the group consisting of: Escherichia, Shigella,    Campylobacter, Burkholderia, Acinetobacter and any combination    thereof.-   66. The animal feed or the animal feed additive according to any one    of items 61 to 65, wherein the Bacillus strain of the animal feed or    the animal feed additive increases the relative abundance of one or    more members of a specific genera selected from the group consisting    of: Ruminococcus, Blautia, Lactobacillus, Faecalibacterium, and    Megasphera in the intestinal microbiome of animals fed with feed    comprising the Bacillus strain compared to animals fed with the same    feed without the Bacillus strain.-   67. The animal feed or the animal feed additive according to any one    of items 61 to 66, wherein lesions in the intestinal tract of an    animal are reduced after feeding the animal feed or the animal feed    additive to the animal for at least 24 hours, such as at least 36    hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week, where the    lesions are reduced compared to lesions in the intestinal tract of    an animal not fed with an animal feed or the animal feed additive    comprising a Bacillus strain.-   68. The animal feed or the animal feed additive according to any one    of items 61 to 67, wherein lesions in the intestinal tract of an    animal are reduced after feeding the animal feed or the animal feed    additive to the animal for at least 2 weeks, such as at least 3    weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6    months or 1 year, where the lesions are reduced compared to lesions    in the intestinal tract of an animal not fed with an animal feed or    the animal feed additive comprising a Bacillus strain.-   69. The animal feed or the animal feed additive according to any one    of items 61 to 68, wherein the Bacillus strain(s) is one or more    Bacillus subtilis strains, one or more Bacillus licheniformis    strains, one or more Bacillus pumilus strains and/or one or more    Bacillus amyloliquefaciens strains.-   70. The animal feed or the animal feed additive according to any one    of items 61 to 69, wherein the Bacillus strain is selected from the    group consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   71. An animal feed or feed additive comprising one or more Bacillus    strains, wherein the Bacillus strain is selected from the group    consisting of:-   Bacillus licheniformis strain O42AH3 having deposit accession number    DSM 32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof,-   Bacillus subtilis strain O52YJ6 having deposit accession number DSM    32560 or a strain having all the identifying characteristics of    Bacillus subtilis strain DSM 32560 or a mutant thereof,-   Bacillus amyloliquefaciens strain O52YYT having deposit accession    number DSM 32561 or a strain having all the identifying    characteristics of Bacillus amyloliquefaciens strain DSM 32561 or a    mutant thereof,-   Bacillus pumilus strain O72NR7 having deposit accession number DSM    32563 or a strain having all the identifying characteristics of    Bacillus pumilus strain DSM 32563 or a mutant thereof, and-   Bacillus subtilis strain SB3175 having deposit accession number NRRL    B-50605 or a strain having all the identifying characteristics of    Bacillus subtilis strain NRRL B-50605 or a mutant thereof.-   72. The animal feed or the animal feed additive according to any one    of items 61 to 71, wherein the Bacillus strain is non-hemolytic.-   73. The animal feed or the animal feed additive according to any one    of items 61 to 72, wherein the Bacillus strain has activity against    Lawsonia intracellularis as determined in Example 4.-   74. The animal feed or the animal feed additive according to any one    of items 61 to 73, wherein the Bacillus strain improves one or more    performance parameters in an animal selected from the list    consisting of body weight gain and/or average daily gain and/or feed    conversion rate in an animal fed with the Bacillus strain.-   75. The animal feed or the animal feed additive according to any one    of items 61 to 74, wherein the animal feed or animal feed additive    is for animals which are selected from the group consisting of:    Horses, guinea pigs, swine, pigs, piglets, growing pigs, sows,    boars, hamsters, and monogastric animals.-   76. The animal feed or the animal feed additive according to any one    of items 61 to 75, wherein the feed or feed additive is for animals    which are selected from the group consisting of: pigs, swine,    piglets, growing pigs, and sows.-   77. The animal feed or the animal feed additive according to any one    of items 61 to 76, wherein the Bacillus strain is the Bacillus    licheniformis strain O42AH3 having deposit accession number DSM    32559 or a strain having all the identifying characteristics of    Bacillus licheniformis strain DSM 32559 or a mutant thereof.-   78. The animal feed or the animal feed additive according to any one    of items 61 to 77, wherein the Bacillus strain is the Bacillus    subtilis strain O52YJ6 having deposit accession number DSM 32560 or    a strain having all the identifying characteristics of Bacillus    subtilis strain DSM 32560 or a mutant thereof.-   79. The animal feed or the animal feed additive according to any one    of items 61 to 78, wherein the Bacillus strain is the Bacillus    amyloliquefaciens strain O52YYT having deposit accession number DSM    32561 or a strain having all the identifying characteristics of    Bacillus amyloliquefaciens strain DSM 32561 or a mutant thereof.-   80. The animal feed or the animal feed additive according to any one    of items 61 to 79, wherein the Bacillus strain is the Bacillus    pumilus strain O72NR7 having deposit accession number DSM 32563 or a    strain having all the identifying characteristics of Bacillus    pumilus strain DSM 32563 or a mutant thereof.-   81. The animal feed or the animal feed additive according to any one    of items 61 to 80, wherein the Bacillus spores of the animal feed or    the animal feed additive are present as dried spores.-   82. The animal feed or the animal feed additive according to any one    of items 61 to 81, which further comprises a carrier.-   83. The animal feed or the animal feed additive according to item    82, wherein the carrier comprises one or more of the following    compounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or    1,3-propylene glycol, sodium chloride, sodium benzoate, potassium    sorbate, sodium sulfate, potassium sulfate, magnesium sulfate,    sodium thiosulfate, calcium carbonate, sodium citrate, dextrin,    maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour,    wheat bran, corn gluten meal, starch and cellulose.-   84. The animal feed or the animal feed additive according to any one    of items 61 to 83, wherein the animal feed or animal feed additive    comprises from 10⁵ to 10¹² CFU/g of isolated Bacillus spores.-   85. The animal feed or animal feed additive according to any one of    items 61 to 84, wherein at least 70% (such as at least 80% or at    least 90%) of the Bacillus spores survive the gastric environment in    a swine such as e.g. pigs, piglets, growing pigs, or sows.-   86. The animal feed or animal feed additive according to any one of    items 61 to 85 which further comprises one or more components    selected from the list consisting of:    -   one or more enzymes;    -   one or more additional microbes;    -   one or more vitamins;    -   one or more minerals;    -   one or more amino acids; and    -   one or more other feed ingredients.-   87. The animal feed or animal feed additive according to any one of    items 61 to 86, wherein the bacterial count of each Bacillus spore    is 1x10⁴ and 1x10¹⁴ CFU/kg of animal feed or animal feed additive,    preferably between 1x10⁶ and 1x10¹² CFU/kg of animal feed or animal    feed additive, and more preferably between 1x10⁷ and 1x10¹¹ CFU/kg    of animal feed or animal feed additive.-   88. The animal feed or animal feed additive according to any one of    items 61 to 87, wherein the animal feed or animal feed additive is a    swine feed or swine feed additive.-   89. The animal feed or animal feed additive according to any one of    items 61 to 88, wherein the animal feed or animal feed additive is    for pigs, swine, piglets, growing pigs, or sows.-   90. A method for improving one or more performance parameter(s) in    an animal comprising the step of administering one or more Bacillus    strains according to any of items 1-35 in the feed of the animal.-   91. A method of inhibiting Lawsonia intracellularis infection in an    animal comprising: administering an effective amount of the Bacillus    strain compositions according to anyone of items 36 to 48 to an    animal such as a swine in need thereof.-   92. A method of preventing development of severe diarrhea in an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to an animal such    as a swine in need thereof for at least 24 hours, such as at least    36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week.-   93. A method of preventing development of severe diarrhea in an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to an animal such    as a swine in need thereof for at least 2 weeks, such as at least 3    weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6    months or 1 year of feeding the Bacillus spore to the animal.-   94. A method of decreasing shedding of Lawsonia intracellularis in    feces of an animal comprising feeding an effective amount of the    Bacillus strain composition according to anyone of items 36 to 48 to    an animal such as a swine in need thereof, where the shedding is    decreased compared to shedding of Lawsonia intracellularis in feces    of animals not fed with Bacillus strains.-   95. A method according to item 94, wherein the Bacillus strain    composition is fed to the animal for at least 24 hours, such as at    least 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week.-   96. A method according to item 94, wherein the Bacillus strain    composition is fed to the animal for at least 2 weeks, such as at    least 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5    months, 6 months or 1 year.-   97. A method of reducing inflammation in an animal comprising    feeding an effective amount of the Bacillus strain composition    according to anyone of items 36 to 48 to an animal such as a swine    in need thereof, wherein the Bacillus strain of the composition    reduces the effect of inflammation on Electrical Resistance in    Caco-2 cells in vitro compared to the effect of inflammation on    Electrical Resistance in Caco-2 cells in vitro without the Bacillus    strain, wherein the effect of inflammation on Electrical Resistance    is measured in a trans-epithelial electrical resistance (TEER) test.-   98. A method of decreasing the relative abundance of one or more    members of the phylum Proteobacteria in the intestinal microbiome of    animals comprising feeding an effective amount of the Bacillus    strain according to anyone of items 36 to 48 to the animal such as a    swine in need thereof for at least 24 hours, such as at least 36    hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week, where the    relative abundance of one or more members of the phylum    Proteobacteria is increased compared to the relative abundance of    Proteobacteria in animals fed with the same feed for the same period    of time without the Bacillus strain.-   99. A method of decreasing the relative abundance of one or more    members of the phylum Proteobacteria in the intestinal microbiome of    animals comprising feeding an effective amount of the Bacillus    strain according to anyone of items 36 to 48 to the animal such as a    swine in need thereof for at least 2 weeks, such as at least 3    weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6    months or 1 year, where the relative abundance of one or more    members of the phylum Proteobacteria is increased compared to the    relative abundance of Proteobacteria in animals fed with the same    feed for the same period of time without the Bacillus strain.-   100. The method according to item 98 or 99, wherein the one or more    members of the phylum Proteobacteria is selected from the group    consisting of: Escherichia, Shigella, Campylobacter, Burkholderia,    Acinetobacter and any combination thereof.-   101. A method of increasing the relative abundance of one or more    members of a specific genera selected from the group consisting of:    Ruminococcus, Blautia, Lactobacillus, Faecalibacterium, and    Megasphera in the intestinal microbiome of animals comprising    feeding an effective amount of the Bacillus strain according to    anyone of items 36 to 48 to the animal such as a swine in need    thereof for at least 24 hours, such as at least 36 hours, 2 days, 3    days, 4 days, 5 days, 6 days or 1 week, where the relative abundance    of one or more members of a specific genera selected from the group    consisting of: Ruminococcus, Blautia, Lactobacillus,    Faecalibacterium, and Megasphera is increased compared to the    relative abundance of the same specific genera in the intestinal    microbiome of animals fed with the same feed for the same period of    time without the Bacillus strain.-   102. A method of increasing the relative abundance of one or more    members of a specific genera selected from the group consisting of:    Ruminococcus, Blautia, Lactobacillus, Faecalibacterium, and    Megasphera in the intestinal microbiome of animals comprising    feeding an effective amount of the Bacillus strain according to    anyone of items 36 to 48 to the animal such as a swine in need    thereof for at least 2 weeks, such as at least 3 weeks, 4 weeks, 1    month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year,    where the relative abundance of one or more members of a specific    genera selected from the group consisting of: Ruminococcus, Blautia,    Lactobacillus, Faecalibacterium, and Megasphera is increased    compared to the relative abundance of the same specific genera in    the intestinal microbiome of animals fed with the same feed for the    same period of time without the Bacillus strain.-   103. A method of reducing lesions in the intestinal tract of an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to an animal such    as a swine in need thereof for at least 24 hours, such as at least    36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week, where    the lesions are reduced compared to lesions in the intestinal tract    of animals not fed with Bacillus strains.-   104. A method of reducing lesions in the intestinal tract of an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to an animal such    as a swine in need thereof for at least 2 weeks, such as at least 3    weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6    months or 1 year, where the lesions are reduced compared to lesions    in the intestinal tract of an animal not fed with Bacillus strains.-   105. A method of reducing the IgG score in an animal comprising    feeding an effective amount of the Bacillus strain composition    according to anyone of items 36 to 48 to said animal such as a swine    in need thereof for at least 24 hours, such as at least 36 hours, 2    days, 3 days, 4 days, 5 days, 6 days or 1 week, where the IgG score    is reduced compared to the IgG score in animals not fed with    Bacillus strains.-   106. A method of reducing the IgG score in an animal comprising    feeding an effective amount of the Bacillus strain composition    according to anyone of items 36 to 48 to said animal such as a swine    in need thereof for at least 2 weeks, such as at least 3 weeks, 4    weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or    1 year, where the IgG score is reduced compared to the IgG score in    animals not fed with Bacillus strains.-   107. A method of increasing the Average Daily Gain of an animal    comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to said animal    such as a swine in need thereof for at least 24 hours, such as at    least 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week,    where the average Daily gain is increased compared to the Average    Daily Gain of animals not fed with Bacillus strains.-   108. A method of increasing the Average Daily Gain of an animal    comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to said animal    such as a swine in need thereof for at least 2 weeks, such as at    least 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5    months, 6 months or 1 year, where the average Daily gain is    increased compared to the Average Daily Gain of animals not fed with    Bacillus strains.-   109. A method of improving the Feed Conversion Rate (FCR) of an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to said animal    such as a swine in need thereof for at least 24 hours, such as at    least 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week,    where the FCR is improved compared to the FCR of animals not fed    with Bacillus strains.-   110. A method of improving the Feed Conversion Rate (FCR) of an    animal comprising feeding an effective amount of the Bacillus strain    composition according to anyone of items 36 to 48 to said animal    such as a swine in need thereof for at least 2 weeks, such as at    least 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5    months, 6 months or 1 year, where the FCR is improved compared to    the FCR of animals not fed with Bacillus strains.-   111. A method of reducing the risk of developing Proliferative    Hemorrhagic Enteropathy (PHE) in an animal comprising feeding an    effective amount of the Bacillus strain composition according to    anyone of items 36 to 48 to said animal such as a swine in need    thereof for at least 24 hours, such as at least 36 hours, 2 days, 3    days, 4 days, 5 days, 6 days or 1 week, where the risk of developing    PHE is improved compared to the same risk in animals not fed with    Bacillus strains.-   112. A method of reducing the risk of developing Proliferative    Hemorrhagic Enteropathy (PHE) in an animal comprising feeding an    effective amount of the Bacillus strain composition according to    anyone of items 36 to 48 to said animal such as a swine in need    thereof for at least 2 weeks, such as at least 3 weeks, 4 weeks, 1    month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year,    where the risk of developing PHE is improved compared to the same    risk in animals not fed with Bacillus strains.

EXAMPLES Example 1 - Deposit of Biological Material

Bacillus licheniformis strain O42AH3 was deposited on Jul. 11, 2017under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure atthe Leibniz-Institut, Deutsche Sammlung von Mikroorganismen undZellkurturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany. Thestrain was designated deposit accession number DSM 32559 on Jul. 13,2017. The strain was deposited under conditions that assure access tothe cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Each deposit represents a pure or substantially pure culture ofthe deposited strain. Each deposit is available as required by foreignpatent laws in countries wherein counterparts of the subject applicationor its progeny are filed. However, it is to be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Bacillus subtilis strain O52YJ6 was deposited on Jul. 11, 2017 under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure at theLeibniz-Institut, Deutsche Sammlung von Mikroorganismen und ZellkurturenGmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany. The strain wasdesignated deposit accession number DSM 32560 on Jul. 13, 2017. Thestrain was deposited under conditions that assure access to the cultureswill be available during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Each depositrepresents a pure or substantially pure culture of the deposited strain.Each deposit is available as required by foreign patent laws incountries wherein counterparts of the subject application or its progenyare filed. However, it is to be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Bacillus amyloliquefaciens strain O52YYT was deposited on Jul. 11, 2017under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure atthe Leibniz-Institut, Deutsche Sammlung von Mikroorganismen undZellkurturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany. Thestrain was designated deposit accession number DSM 32561 on Jul. 13,2017. The strain was deposited under conditions that assure access tothe cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Each deposit represents a pure or substantially pure culture ofthe deposited strain. Each deposit is available as required by foreignpatent laws in countries wherein counterparts of the subject applicationor its progeny are filed. However, it is to be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Bacillus subtilis strain O52YZ6 was deposited on Jul. 11, 2017 under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure at theLeibniz-Institut, Deutsche Sammlung von Mikroorganismen und ZellkurturenGmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany. The strain wasdesignated deposit accession number DSM 32562 on Jul. 13, 2017. Thestrain was deposited under conditions that assure access to the cultureswill be available during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Each depositrepresents a pure or substantially pure culture of the deposited strain.Each deposit is available as required by foreign patent laws incountries wherein counterparts of the subject application or its progenyare filed. However, it is to be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Bacillus pumilis strain O72NR7 was deposited on Jul. 11, 2017 under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure at theLeibniz-Institut, Deutsche Sammlung von Mikroorganismen und ZellkurturenGmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany. The strain wasdesignated deposit accession number DSM 32563 on Jul. 13, 2017. Thestrain was deposited under conditions that assure access to the cultureswill be available during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Each depositrepresents a pure or substantially pure culture of the deposited strain.Each deposit is available as required by foreign patent laws incountries wherein counterparts of the subject application or its progenyare filed. However, it is to be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Bacillus subtilis strain SB3175 was deposited on Nov. 30, 2011 under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure at theAgricultural Research Service Culture Collection, 1815 North UniversityStreet, Peoria, Illinois 61604, U.S.A. The strain was designated depositaccession number NRRL B-50605. The strain was deposited under conditionsthat assure access to the cultures will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and35 U.S.C. §122. Each deposit represents a pure or substantially pureculture of the deposited strain. Each deposit is available as requiredby foreign patent laws in countries wherein counterparts of the subjectapplication or its progeny are filed. However, it is to be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Sequencing of 16S rDNA Genes

DNA was extracted from a culture of DSM 32559, DSM 32560, DSM 32561 orDSM 32563 using QiaAmp DNA Blood Mini Kit (Qiagen art 51106). The kitwas used as recommended for extraction of DNA from gram positivebacteria.

16S rDNA was amplified in a total volume of 50 µl by mixing: 10 pmol ofeach of Primer 16S F and 16S R, 0.2 mM of each nucleotide, 2.5 unitsAmpli taq, 1 x Ampli taq buffer, 5 µl DNA template and by using thefollowing PCR program: 94° C. 2 min (94° C. 30 s, 52° C. 30 S, 72° C. 1min)x35, 72° C. 10 min on a Perkin Elmer PCR machine. The PCR productwas sequenced by Novozymes DNA sequencing facility using primer 530R,357F, 1390R and 1100F.

TABLE 1 Primers Primer Sequence SEQ ID NO 16S-F 5′-GAGTTTGATCCTGGCTCAG-3′ SEQ ID NO: 5 16S-R 5′ -AGAAAGGAGGTGATCCAGCC-3′SEQ ID NO: 6 794-R 5′ -ATCTAATCCTGTTTGCTCCCC-3′ SEQ ID NO: 7 357-F 5′-TACGGGAGGCAGCAG-3′ SEQ ID NO: 8 1390-R 5′ -CGGTGTGTRCAAGGCCC-3′ SEQ IDNO: 9 1000-F 5′ -CAACGAGCGCAACCCT′, SEQ ID NO: 10

Degeneration of primer 1390-R: R is A or G. The 16 S rDNA sequences fromDSM 32559, DSM 32560, DSM 32561, DSM 32563, O52YZ5 and O22FHD are shownas SEQ ID NO: 1-4 and 11-12 in the sequence listing respectively. The 16S rDNA sequences from DSM 32559, DSM 32560, DSM 32561, DSM 32563, O52YZ5and O22FHD were analyzed by BLAST against EMBL database and showedidentity to 16 S rDNA sequences of respectively Bacillus y (SEQ ID NO:1), Bacillus subtilis (SEQ ID NO: 2), Bacillus amyloliquefaciens (SEQ IDNO: 3), Bacillus pumilus (SEQ ID NO: 4), Bacillus subtilis (SEQ ID NO:11) and Bacillus subtilis (SEQ ID NO: 12).

In order to study the phylogenetic affiliation of SEQ ID NO: 1 to SEQ IDNO: 4 and SEQ ID NO: 11 to SEQ ID NO: 12, the sequences were analyzed bya ClustalW alignment in MegAlign (DNASTAR) using SEQ ID NO: 5 to SEQ IDNO: 7 as benchmark. These sequences are reference 16S rDNA sequences ofthe type strains of Bacillus vallismortis taken from AB021198 (SEQ IDNO: 5), Bacillus subtilis taken from AJ276351 (SEQ ID NO: 6) andBacillus amyloliquefaciens taken from AB255669 (SEQ ID NO: 7).

The ClustalW alignment of SEQ ID NO: 1 to SEQ ID NO: 7 shows nucleotidepositions where 2 or more sequences have a nucleotide that deviates fromthe other.

Example 2 - Cell-culture Assay Test of Cell-free Extracts of Bacillusfor Reduction in Heavy Infected Cells (HIC) Preparation of TSBYE pH 6.2Media

Tryptic Soy Broth (30 g/L) was mixed with Yeast Extract (6 g/L) andwater pH was adjusted to 6.2 using HCl. The resulting TSBYE media wasautoclaved before use.

Preparation of Cell-Free Bacillus Extracts

Bacillus extracts were prepared over three days. On day 1 of theexperiment, sterile culture tubes were prepared with 10 ml of Trypticsoy broth with 0.6% yeast extract (TSBYE) at pH 6.2. The culture tubeswere inoculated so that each tube contained a single colony from a plateof a Bacillus strain. A separate culture tube was inoculated with aBSL-2 pathogenic isolate of E. coli (ATCC10536). Both the Bacillusstrains and the E. coli were incubated overnight at 35° C., with 200 rpmshaking in atmospheric oxygen. On day 2 of the experiment, 1 ml of E.coli culture was aliquoted into a microfuge tube. E. coli was heatkilled in a 80° C. water bath for 30 minutes. After the heat kill, asample from the microfuge tube was streaked onto SMA plates to ensureall E. coli was heat killed. The plate was incubated overnight at 35° C.Sterile culture tubes were prepared with 9 ml of TSBYE pH 6.2 media, 1ml of heat killed E. coli was added to each culture tube, and the tubewas inoculated with 100 ul from the Bacillus culture prepared on day 1.The cultures were incubated overnight at 35° C. with 200 rpm shaking. Onday 3 of the experiment, the primed Bacillus overnight cultures weretransferred to 15 ml conical tubes. The cells were centrifuged at 8,000rpm for 10 min and the supernatant was filtered two times through a 0.22uM filter into a clean, sterile conical tube for use in the in vitroassay for activity against Lawsonia intracellularis. A sample offiltered supernatant was streaked onto SMA plates to ensure that allcells had been filtered out. The supernatant was incubated overnight at35° C. and checked for lack of growth.

Testing of Cell-Free Extracts of Bacillus for Activity Against LawsoniaIntracellularis

Bacillus cell-free extracts were evaluated in vitro for activity againstLawsonia intracellularis strain GBI06, a recent pathogenic isolateprovided by Gut Bugs. Extracts taken from Bacillus strains wereconsidered positive if they could reduce the re-infection ability ofLawsonia intracellularis by at least 30% following co-incubation.Reduction in Lawsonia intracellularis ability in vitro was calculated bycomparison of the level of heavily-infected cells (HIC) in theBacillus-extract treated group compared to the control group that didnot contain a Bacillus extract. The in vitro Minimum InhibitoryConcentration (MIC) of the Bacillus cell-free extracts were measured toassess in vivo sensitivity in a porcine challenge model.

The pathogenic Lawsonia intracellularis isolate, GBI06, was collectedfrom a field case in 2006. This strain was grown in a murine fibroblastMcCoy cell (CRL 1696, American Type Culture Collection, Virginia US).GBI06 was maintained in a cell culture system as described previously(Guedes and Gebhart, 2003, Comparison of intestinal mucosa homogenateand pure culture of the homologous Lawsonia intracellularis isolate inreproducing proliferative enteropathy in swine. Vet. Microbiol. 93,159-166). The isolate was cultured in a way that demonstratedintracellular as well as extracellular MIC data. The tissue culturesystem used to demonstrate antimicrobial sensitivity was based on aprevious study (Wattanaphansak et al., 2008, Development and validationof an enzyme-linked immunosorbent assay for the diagnosis of porcineproliferative enteropathy. J. Vet. Diagn. Invest. 20, 170-177).

Antimicrobials

Tylosin tartrate (Sigma-Aldrich, Missouri, United States) was used as acontrol during testing. A stock solution of tylosin tartrate wasprepared to a final concentration of 2,560 µg/ml. The tylosin tartratewas dissolved in sterile distilled water. The tylosin tartrate wasfilter sterilized using a 0.2 µm filter and stored at -20° C. untilneeded. Two-fold serial dilutions of the stock solution were preparedand working solutions at the following dilutions 1,280, 640, 320, 160,80, 40, 20, 10, 5, 2.5, and 1.25 µg/ml were produced. The workingsolutions were further diluted 1:10 by adding 10 µl of antimicrobialstock solution into 90 µl of cell culture media to yield finalconcentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125µg/ml.

Extracellular Testing

Extracellular testing was performed to demonstrate the effect particularantimicrobials have on Lawsonia intracellularis prior to entering thehost cell. A 96-well plate as shown in Table 2 was inoculated withmurine fibroblast McCoy cells and incubated for 24 hours to getapproximately 20% confluency. In a second 96-well plate, 100 µl ofLawsonia intracellularis cell culture media containing approximately 10⁷Lawsonia intracellularis cells was added to each well. To make dilutionsof Bacillus supernatants, 100 µl of supernatant was added to the firstrow of media containing Lawsonia intracellularis. This resulted in thefirst 1:2 dilution, dilutions were continued 1:2 down the plate to thehighest dilution 1:16. Each supernatant dilution series were made induplicate per plate. Appropriate duplicate tylosin tartrate dilutionswere made in duplicate on each plate to achieve the following dilutions(0.125 µg/mL, 0.25 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL, 4 µg/mL, 8 µg/mL,16 µg/mL, 32 µg/mL, 64 µg/mL, and 128 µg/ml). The plates containingdilutions were then incubated at 37° C. with atmospheric conditions of8.0% oxygen, 8.8% carbon dioxide, and 83.2% nitrogen for 2 hours. Thesebacterial suspensions were then placed onto the McCoy host cells thatwere seeded 24 hours prior, and allowed to incubate for another 24hours. The 96-well plate was then incubated for another 5 days.

Antimicrobial Dilution Supernatant Dilution Bacillus Strain XYZ BacillusStrain XYZ Bacillus Strain XYZ Bacillus Strain XYZ Lawsonia ControlTylosin Tartrate Tylosin Dilution 1:2 1:128 1:4 1:64 1:8 1:32 1:16 1:161:2 1:8 1:4 1:4 1:8 1:2 1:16 1:1

Table 2 contains a schematic diagram of how plates are organized forintracellular and extracellular assays.

Calculating MIC Value

After the 96-well plates had incubated for 5 days, the supernatant fromthe infected plates was removed and the cell culture monolayer was fixedwith 100 µl of a cold solution containing 50% acetone and 50% methanolfor 1 minute. To assess the inhibitory effect of each antimicrobial onLawsonia intracellularis proliferation, the infected plates werestrained using a modified immunoperoxidase monolayer assay stainingmethod (Guedes et al., 2002, Validation of an immunoperoxidase monolayerassay as a serologic test for porcine proliferative enteropathy. J. Vet.Diagn. Invest. 14, 528-530).

The Lawsonia intracellularis proliferation was evaluated by counting thenumber of Heavily Infected Cells (HIC) (McOrist et al., 1995,Characterization of Lawsonia intracellularis gen. nov, sp nov, theobligately intracellular bacterium of porcine proliferative enteropathy.Int. J. Syst. Bacteriol. 45, 520-525) in each well using an invertedmicroscope. Host cells were considered to be HIC if the number ofintracellular Lawsonia intracellularis had proliferated to greater than30 Lawsonia intracellularis bacteria per host cell. The number of HICsin each well was compared as a percentage of the average HICs of thecontrol (non-treated) wells. Cell-free Bacillus extracts were consideredto have activity against Lawsonia intracellularis in vitro if theyshowed 30% or greater reduction in the number of HICs compared tountreated controls.

Results

Seven different Bacillus extracts were identified that had a positiveinhibitory effect on Lawsonia infection of cells when tested asdescribed above. Out of these, six extracts were retested for activityagainst Lawsonia intracellularis and four out of six extracts hadrepeatable activity against Lawsonia intracellularis.

TABLE 3 Strain ID Bacillus Species In vitro Activity Against LawsoniaO52YJ6 B. subtilis Yes O52YZ5 B. subtilis Yes O52YYT B.amyloliquefaciens Yes O22FHD B. subtilis Yes O72NR7 B. pumilus YesO42AH3 B. licheniformis Yes SB3175 B. subtilis Yes NRRL B-50015 B.licheniformis No

Example 3 - Effect on Fecal Score in Piglets Orally Challenged WithLawsonia Intracellularis

On day -3 of the experiment, 125 three-week old piglets were randomizedby weight and gender and divided into 25 pens of 5 animals each. The 25pens were randomized into 5 treatment groups with 5 pens each. Animalsin three groups were then put on a standard diet, while animals inanother group were put on the standard diet to which 1x10¹²colony-forming units (CFU) of spores of Bacillus subtilis O52YJ6 hadbeen added (O52YJ6 group). Animals in the remaining group received thesame diet as in group O52YJ6 except that spores of Bacillus pumilus 3002were used instead of B. subtilis O52YJ6 (group 3002). All diets were fedthroughout the entire experiment to the same animals.

On day 0, all animals from one group on the standard diet werevaccinated orally with the Enterisol vaccine from Boehringer Ingelheimaccording to the manufacturer’s instructions (vaccine or VAC group).Another group on the standard diet did not receive any further treatmentor challenge (negative control or NC group). The remaining group will bereferred to as positive control or PC group.

On day 21, all animals, except those in the NC group, received anintragastric gavage with a homogenate of pig intestinal mucosal tissuethat contained large numbers of the intracellular pathogenic bacteriumLawsonia intracellularis, the causal agent of proliferative enteropathy(PE).

On day 42 all animals were euthanized and examined for signs of PE.Fecal appearances were scored per pen on days 20, 28, 31, 35, 38. Foreach pen, a score of 0 was attributed when the majority of fecal matterhad a normal appearance. A score of 1 was attributed when fecal matterwas mainly soft, with a cow-pie consistency. A score of 2 meant thatfecal matter was moderately loose and fell through the slats of thefloor. A score of 3 was assigned when the majority of fecal matter waswatery, possibly blood-tinged, possible necrotic debris.

Blood samples were taken from each animal on days 0, 20 and 42.

TABLE 4 Xhi-square test of cumulative fecal scorings in each group. A <symbol indicates that a treatment had less frequently a certain scorethan could be expected if none of the treatments had any effect. Thesign > indicates increase in occurrence. Table 4 shows that animals inthe O52YJ6 group had less often a score of 2 or 3 than animals in the PCgroup. 0 1 2 or 3 PC < > > VAC > < < O52YJ6 < > < 3002 < > > NC > < <

Results

As shown in FIG. 1 , animals in group 1 produced mainly fecal matterover the course of the experiment that was of normal appearance (score0). Only 3% of cases had a score higher than 0. Animals in the PC grouphad the most often scores superior to 0. A Chi square test, of which theresults are in table 4, indicates that animals in the O52YJ6 group hadless often a fecal score of “3” than animals in the PC group, suggestingthat ingestion of Bacillus subtilis O52YJ6 could protect animals fromdeveloping severe diarrhea upon challenge with Lawsonia intracellularis.

Example 4 - Effect on Growth and Immune Responses in Piglets OrallyChallenged With Lawsonia Intracellularis General

A total of 60 newly weaned piglets (age 21 days) was randomized byweight and gender over 5 treatments groups, as follows:

-   1. Control group (N=10). Animals received standard diet throughout    the experiment and were not challenged with Lawsonia    intracellularis;-   2. Challenged, untreated group (N=20). Animals received standard    diet throughout the experiment and were challenged at age 52 days    via intragastric gavage with Lawsonia intracellularis homogenate    (approximately 2.0x 10⁷ bacteria / dose) diluted into 40 mL of    sterile carrier buffer;-   3. O42AH3 group (N=10). Animals received standard diet to which had    been added 1 x 10^12 CFU of spores of Bacillus licheniformis strain    O42AH3 from weaning till slaughter and were challenged at age 52    days with Lawsonia intracellularis;-   4. O72NR7 group (n=10). As group 3, except that Bacillus pumilus    O72NR7 was used instead of B. licheniformis O42AH3;-   5. O52YYT group (n=10). As group 3, except that Bacillus    amyloliquefacines O52YYT was used instead of B. licheniformis    O42AH3.

Effect On Bodyweight Gain

Bodyweights were individually determined at age 21 days, 52 days(immediately before challenge), and immediately before euthanasia andnecropsy at day 70, and average daily gains (ADG) were determined forthe pre-challenge period, post-challenge period, and entire period, bydividing weight gains (in grams) over the respective periods by thenumber of days in that period.

As seen in FIG. 2 , all animals challenged with Lawsonia intracellularishad a significantly lower ADG post-challenge than animals in group 1(no-challenge;), indicating that the challenge with Lawsoniaintracellularis had, as predicted, a negative impact on growthperformance. However, animals receiving O42AH3 (Group 3) or O52YYT(Group 5) had a higher ADG than challenged animals without treatment(Group 2). For O42AH3 and O52YYT the increases in ADG versus untreatedwere 8.7% and 10.8% respectively. O72NR7 showed an improvement of 0.4%.Taking into account the entire experimental duration, all threeprobiotics had higher ADG than challenged animals with no treatment(2.7, 3.1 and 3.1 % for O42AH3, O72NR7 and O52YYT, respectively).

Lawsonia Intracellularis Infectivity

To test the ability of the Bacillus strains O42AH3, O72NR7 and O52YYT toinhibit infectivity of Lawsonia intracellularis, we measured twoparameters: 1) Levels of Immunoglobulin G (IgG) in the serum of pigletsfrom groups 1 - 5 obtained after slaughter and 2) the number of Lawsoniaintracellularis cells per gram feces obtained at various time pointafter challenge.

Effect On Seroconversion

An ELISA was used to measure IgG against extracts of whole Lawsoniaintracellularis. A readout of 0-20 indicates a negative test; a readoutof 20-30 indicates borderline seropositivity; a readout >30 means theanimal is seropositive for anti-Lawsonia intracellularis IgG and hasdeveloped infection. As shown in FIG. 3 , it was observed that pigletsreceiving a diet containing O42AH3, O72NR7, or O52YYT before fromweaning till slaughter all had lower average numbers of anti-Lawsoniaintracellularis IgG in their serum than animals in group 2, indicatingthat all three Bacillus strains were able to limit infectivity ofLawsonia intracellularis. In FIG. 4 , the results are also expressed asthe fraction of animals per group that is sero-positive, i.e. of whichthe ELISA values exceeded 30.

Effect on Fecal Lawsonia Intracellularis Shedding

Fecal samples were obtained from all animals in all groups, immediatelybefore the oral challenge (post-challenge day 0), and on days 2, 4, 6,8, 10, 12 and 15 post-challenge. DNA was isolated from the feces and theamount of DNA belonging to Lawsonia intracellularis was quantified withreal-time PCR. The use of a standard curve plotting Ct values versusthose obtained with known amounts of DNA allowed us to calculate thenumber of Lawsonia intracellularis bacteria per gram feces. As shown inFIG. 5 , non-challenged animals (Group 1) had no detectable Lawsoniaintracellularis DNA in their feces at any time point, whereas allchallenged animals started to shed significant amounts of Lawsoniaintracellularis at the latest by day 8 post-challenge. Animals in groups3, 4 and 5 shed lower amounts of Lawsonia intracellularis than animalsin group 2 (area under the curves) and the time of onset of shedding wasalso delayed compared to group 2 (FIG. 5 ). Thus, inclusion of one ofeither of the three strains of Bacillus in the diet limited shedding ofLawsonia intracellularis in the feces, suggesting that Lawsoniaintracellularis replication in animals on these diets was impaired.

Pathology

Since piglets with either of the three probiotics in the diet showeddecreased fecal Lawsonia intracellularis shedding and reduced immuneresponses to Lawsonia intracellularis upon Lawsonia intracellularischallenge compared to non-treated challenged animals, we hypothesizedthat these probiotics would also reduce intestinal pathology due toinfection with Lawsonia intracellularis. To this end, macroscopicallyvisible thickening of the small intestine, a hallmark of PE (Vannucci,F. A., & Gebhart, C. J. (2014). Recent advances in understanding thepathogenesis of Lawsonia intracellularis infections. Vet.Pathol., 51,465-477) was scored with a semi-quantitative scale, with “0”representing no thickening and “3” presenting most severe thickening.FIG. 6 represents the distribution of animals with no or only minorthickening (scores 0, 1) versus animals with mild or severe thickening(scores 2, 3) for each group.

The results indicate that all tree probiotics were able to reducesmall-intestinal thickening after the Lawsonia intracellularis challengecompared to untreated, Lawsonia intracellularis -challenged animals.

For each animal also a sum of scores of several microscopic parametersof PE was established. The individual components that were scored wereas follows: Thickening of crypts and villi (0 = no thickening; 3 = worstthickening); microscopic scoring of lymphohistiocytic infiltration (0 =non; 3 = worst); and microscopic assessment of inflammation (0 = non; 3= worst). Animals with a score of 9 had been severely affected, a scoreof 0 indicated absence of signs of disease. Results, shown in FIG. 7 ,showed that all three probiotics resulted in lower small intestinalmicroscopical inflammation upon challenge than untreated challengedanimals.

Example 5 - Effect on Growth and Immune Responses in Piglets NaturallyChallenged With Lawsonia Intracellularis: General:

A total of 1728 weaned piglets, obtained by breeding Large White xLandrace- type sows (Topigs 40) with Duroc-type boars (Topigs Talent)),average age of 28 days, were randomized over 4 treatments.

Treatment 1 (T×1) received a standard diet (mainly wheat, barley andcorn during post-weaning and mainly wheat and corn during fattening).

T×2 received the same diet but piglets were vaccinated with Enterisol(Boehringer Ingelheim) per os at weaning as per the manufacturer’sinstructions.

T×3 and T×4 were not vaccinated and received the same diets as T×1 andT×2, to which was added 1×10¹² CFU / ton NZ014 (T×3) or 1×10¹² CFU / tonO72NR7 (T×4).

Respective diets were administered from weaning till slaughter. Animals,balanced for bodyweight and gender, were randomized to one of these fourtreatment groups spread over 8 rooms in the nursery building and over 4rooms in the fattening building.

Effect on Bodyweight Gain

Bodyweight and feed intake were determined per pen on the start of thestudy (average age: 28 days), at age 42 d, 70 d, 135 d and 165 d.Bodyweights (BW) of deceased animals were noted at the day of death.Average daily gain (“ADG”, in g/day) and feed conversion rate (“FCR”;daily feed intake / ADG) were calculated for the nursery phase as awhole (d28 - d70) or for the fattening phase as a whole (d70-d165).

In FIG. 8 , NC (“negative control”) refers to animals receiving standarddiet; PC to animals on standard diet but who were all vaccinated withEnterisol within a week of weaning; O42AH3 and O72NR7 refers to groupsof animals receiving standard to which the respective Bacillus strainshad been added at an inclusion rate of 1 × 10¹² CFU / ton feed.

As evident from FIG. 8 , compared to untreated control animals (“NC”group), animals vaccinated with Enterisol or animals with either O42AH3or O72NR7 in their diet showed significantly increased average dailygains (ADG) and significantly improved feed conversion rates (FCR) overall time periods tested. From day 28 of age till age 70, considered thenursery stage, ADG improvements of the Enterisol (PC) group, O42AH3group, and O72NR7 group, all versus the negative control (NC) group,were 6.7%, 4.5% and 4.7%, respectively. Corresponding FCR improvementswere 5.2%, 2.5% and 3.7%. From age 70 till age 165, considered thefattening phase in pig production, improvements in ADG versus NC groupswere 6.6% for the PC group, 3.8% for the O42AH3 group, and 3.0% for theO72NR7 group, respectively. Corresponding improvements in FCR were 9.1,5.0, and 2.2%, respectively.

Effect on Development of Anti-Lawsonia Intracellularis IgG

Blood samples were collected at age 70 days from 3 randomly chosen pigsper pen for detection of anti-Lawsonia intracellularis IgG by ELISA(Svanova Bioscreen). An animal’s serum was considered seropositiveaccording to the manufacturer’s instructions.

As shown in figure xxx, the percentage animals in the O42AH3 and theO72NR7 groups testing seropositive for Lawsonia was reduced compared tothe percentage seropositive animals in the NC group. Thus, adding O42AH3or O72NR7 to the diet had reduced the infectivity of Lawsoniaintracellularis.

Example 6 - Effect on Growth and Fecal Shedding in Piglets OrallyChallenged With Lawsonia Intracellularis: General

This trial was conducted on a commercial farm in Korzękwice, Poland,from December 2017 -October 2018. Presence of Lawsonia intracellularison the farm was confirmed by serological analysis.

A total of 768 weaned male and female piglets, average age of 28 days,were randomized over 4 treatments.

Treatment 1 (T×1; negative control “NC”)) received a standard diet(mainly wheat, barley, soy meal).

T×2 (positive control; “PC”) received the same diet but piglets werevaccinated with Enterisol (Boehringer Ingelheim) per os at weaning asper the manufacturer’s instructions.

T×3 and T×4 were not vaccinated and received the same diet as T×1 andT×2 but supplemented with 1×10¹² CFU / ton O52YYT (T×3) or 1×10¹² CFU /ton O72NR7 (T×4), respectively, from weaning till slaughter.

Animals, balanced for bodyweight and gender, were randomized to one ofthese four treatment groups.

Effect on Bodyweight Gain

Bodyweight and feed intake were determined per pen on the start of thestudy (average age 28 days; start of nursery phase), at age 45d, 88d(end of nursery phase), 147d, and 207d. Bodyweights (BW) of deceasedanimals were noted at the day of death. Average daily gain (“ADG”, ing/day or kg/day), average daily feed intake (“FI” in g/day) and feedconversion rate (“FCR”; FI / ADG) were calculated for each period aswell as for the nursery phase as a whole (d28 - d78), for the fatteningphase as a whole (d88-d207), and for the entire post-weaned lifespan ofthe animals.

Results in FIG. 10 show that, on average, animals in the positivecontrol group (PC; all animals in this group were vaccinated withEnterisol within a week from weaning) showed improved ADG and FCRcompared with animals in the negative control group (NC) for all timeperiods tested, including the entire nursery phase (age day 28 - day 88)and the entire fattening phase (age day 88 days - 207). Animals withO72NR7 in their diet also had significantly improved ADG and FCR in thenursery phase and in the fattening phase compared to animals in the NCgroup. The same was observed for animals with O52YYT in their diet.

Effect on Fecal Lawsonia Intracellularis Shedding

Rectal fecal samples were obtained from two randomly selected animalsper pen at day 88 and 147 and Lawsonia intracellularis DNA in thesamples was identified by realtime PCR using the “Amplitest” Lawsoniaintracellularis realtime PCR assay from Amplicon Inc. (Wroclaw, Poland).

As shown in FIG. 11 , animals vaccinated with Enterisol (PC group) oranimals receiving either O72NR7 or O52YYT in their diet shedsignificantly less Lawsonia intracellularis in their feces, indicatingthat dietary enrichment with O72NR7 or O52YYT reduced the presence ofLawsonia intracellularis in their digestive tract.

Example 7: Trans-epithelial Electrical Resistance of Bacillus

Trans-epithelial electrical resistance (TEER) was used as an in vitromodel for the strength of the intestinal barrier function. The greaterthe electrical resistance, the stronger the barrier function.

Method

In a transwell system, polarized Caco-2 cells were stimulated witheither bacteria alone, inflammation conditions alone (TNFalpha,INFgamma), or with both bacteria and inflammation. TEER was recordedevery hour over a period of 48 hours. Data was normalized to the averageof 6 hours prior to stimulation. Technical duplicates were performed perexperiment.

Results:

O72NR7 (DSM 32563) caused a prolonged increase in TEER values,indicating a strengthening of the barrier function in vitro, compared tocontrol cells (FIG. 12 ). Inflammatory conditions often cause a drop inTEER, indicating a weakened barrier function. With O72NR7 (DSM 32563)tested under inflammatory conditions, the TEER values were rescuedcompared to the control or the control + inflammation groups (FIG. 13 ).

Example 8: Microbiome Sequencing and Analysis Sample Collection

Rectal swabs were collected from a single animal per pen and preservedin sterile phosphate buffer. After soaking, swabs were discarded and theremaining material dissolved in phosphate buffer frozen for storage at-80° C. Material was later used for DNA extractions. A total of 96samples were collected.

DNA Extractions Using the Powerlyzer Powersoil DNA Extraction Kit

250 ul of the buffer from the swab sample was placed into a PowerBeadTube and 750 ul of Powerbead Solution was added to the PowerBead Tube.60 ul of Solution C1 was added and inverted several times or vortexedbriefly. The PowerBead Tubes were secured onto the FastPrep system andshook at 1600 rpm for 60 seconds. The bead tubes were centrifuged at10,000 rcf for 1 minute. 450 ul of the supernatant were transferred to aclean 2 ml Qiacube collection cuvette and the Qiacube was set upaccording the Protocol instructions for Powerlyzer Powersoil kit and theprotocol was run accordingly. Once extraction was complete, DNA sampleswere frozen in cryovials and stored at -80° C. until ready to use.

Sequencing and Analysis

DNA extraction, PCR amplification of the 16S RNA gene and libraryconstruction: PCR amplification was done according to Phusion.Pcrl with20 cycles. DNA was measured using the HS kit for quantification.Post-PCR cleanup was done according to the protocol below.

Montage PCR clean up: 5 ul from each PCR were removed and pooled in aDNA LowBind Eppendorf tube. 300 µl of the pooled sample was added on thefilter part of Montage tubes and centrifuged in Montage tubes at 3000 Gfor 25 min. The Montage tube with supernatant was discarded. The filterpart of the Montage tubes was kept and placed in new Montage tubes. 20µl of elution buffer-10 mM Tris-HCl pH 8 was added on the filter part ofthe Montage tubes and mixed using a pipette. The filter part was turnedupside-down in new Montage tubes, then the sample was centrifuged inMontage tubes at 2000 G for 5 min. Finally, the supernatant wastransferred to DNA LowBind Eppendorf tubes.

Bioinformatics Processing, OTU Clustering and Classification

The generation of Operational Taxonomic Unit (OTU) tables was done withusearch version 10.0.240 (UNOISE2: improved error-correction forIllumina 16S and ITS amplicon sequencing:www.biorxiv.org/content/early/2016/10/15/081257). Primer binding regionswere removed with fastx_truncate and reads were filtered to contain lessthan one error per read. The quality filtered reads were denoised withunoise3. OTU abundance was calculated by mapping with usearch_globalusing a 97% identity threshold. Taxonomical classification was done withthe RDP classifier version 2.12. The phylogentic tree was made byaligning the 16S sequences with mafft and the tree was inferred byFastTree.

Statistical Analysis

The results were analyzed in R using the ampvis package v.1.9.1(Albertsen et al., 2015, Back to basics the influence of DNA extractionand primer choice on phylogenetic analysis of activated sludgecommunities. PLoS One. 2015 Jul 16;10), which builds on the R packageDESeq2 (Love et al., 2014. Moderated estimation of fold change anddispersion for RNA-seq data with DESeq2, Genome Biology 15(12): 550) fordetecting species in differential abundance. Beta diversity was analyzedby calculating Unifrac distances. Variance analysis of beta diversitywas done with adonis (Permutational multivariate analysis) from thevegan package.

Results

Animals in both the Bacillus licheniformis strain O42AH3 and Bacilluspumilus strain O72NR7-fed groups showed several changes to theintestinal bacterial community as determined by comparisons of relativeabundance. Here, relative abundance indicates the proportion ofindividual sequence reads in an individual animal sample thatcorresponds to a known and identified taxonomic group, compared to allsequence reads in the same sample. In both O42AH3 and O72NR7-fed animalsthere was a reduction in the overall level of members of the phylumProteobacteria, compared to controls (Table 5).

TABLE 5 Relative abundance, expressed as a percentage, of the phylumProteobacteria in different treatment groups Treatment Percentage ofrelative abundance of Proteobacteria Control 14.1 Enterisol vaccine 5.5Bacillus licheniformis strain O42AH3 4.1 Bacillus pumilus strain O72NR72.3

Within the phylum Proteobacteria, several genera were reduced in bothO42AH3 and O72NR7-fed animals, compared to the control. These includedthe genus Escherichia/Shigella, Campylobacter, Burkholderia, andAcinetobacter (Table 6).

TABLE 6 In the phylum Proteobacteria, several specific genera hadreduced relative abundance in Bacillus-fed treatment groups compared tothe control or Enterisol Treatment Escherichia/Shigella CampylobacterBurkholderia Acinetobacter Control 4.2 3 2.1 10.1 Enterisol 3.8 3.7 2 0042AH3 2.2 2.5 1.6 0.1 O72NR7 2 0.7 0.7 0.2

Additionally, several members of the intestinal community had higherrelative abundance in O42AH3 and/or O72NR7-fed animals, includingmembers of the genus Ruminococcus, Blautia, Lactobacillus, Megasphaeraand Faecalibacterium (Table 7).

TABLE 7 Some specific genera were increased in relative abundance inBacillus-fed treatment groups compared to the control or EnterisolTreatment Lactobacillus Megasphaera Faecalibacterium RuminococcusBlautia Control 10.1 0.3 0.6 0.7 0.5 Enterisol 31.1 1.2 1.3 1.2 0.7O42AH3 20.5 2.6 1.5 1.2 0.7 O72NR7 34 3.7 2.9 1.4 1.3

Example 9: Animal Feed and Animal Feed Additives Comprising a BacillusStrain Animal Feed Additive

A formulation comprising the Bacillus strain of the invention (e.g.Bacillus licheniformis O42AH3 (deposit no. DSM 32559), Bacillus subtilisO52YJ6 (deposit no. DSM 32560), Bacillus amyloliquefaciens O52YYT(deposit no. DSM 32561), or Bacillus pumilus O72NR7 (deposit no. DSM32563) containing 0.01 g to 10 g Bacillus strain is added to thefollowing premix (per kilo of premix):

-   5000000 IE Vitamin A-   1000000 IE Vitamin D3-   13333 mg Vitamin E-   1000 mg Vitamin K3-   750 mg Vitamin B1-   2500 mg Vitamin B2-   1500 mg Vitamin B6-   7666 mcg Vitamin B12-   12333 mg Niacin-   33333 mcg Biotin-   300 mg Folic Acid-   3000 mg Ca-D-Panthothenate-   1666 mg Cu-   16666 mg Fe-   16666 mg Zn-   23333 mg Mn-   133 mg Co-   66 mg I-   66 mg Se-   5.8% Calcium-   25% Sodium

Animal Feed

This is an example of an animal feed for swine comprising the animalfeed additive as described above:

-   36.425% Barley-   30.000% Wheat-   12.000% Rapeseed meal-   12.789% Soybean meal-   5.457% Animal fat-   0.975% Calcium carbonate-   0.742% Monocalcium phosphate-   0.408% Salt-   0.096% Methionine-OH-   0.609% L-lysine (50)-   0.099% L-threonine-   0.400% Vitamin&mineral premix

The ingredients are mixed, and the feed is pelleted at the desiredtemperature, e.g. 60, 65, 75, 80, 85, 90 or even 95° C.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

That which is claimed:
 1. A composition comprising calcium carbonate andone or more Bacillus strains selected from the group consisting of:Bacillus licheniformis strain O42AH3 having deposit accession number DSM32559 and strains having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559, Bacillus subtilis strain O52YJ6 havingdeposit accession number DSM 32560 and strains having all theidentifying characteristics of Bacillus subtilis strain DSM 32560,Bacillus amyloliquefaciens strain O52YYT having deposit accession numberDSM 32561 and strains having all the identifying characteristics ofBacillus amyloliquefaciens strain DSM 32561, and Bacillus pumilus strainO72NR7 having deposit accession number DSM 32563 and strains having allthe identifying characteristics of Bacillus pumilus strain DSM
 32563. 2.The composition of claim 1, comprising Bacillus licheniformis strainO42AH3 having deposit accession number DSM 32559 and/or a strain havingall the identifying characteristics of Bacillus licheniformis strain DSM32559.
 3. The composition of claim 1, comprising Bacillus subtilisstrain O52YJ6 having deposit accession number DSM 32560 and/or a strainhaving all the identifying characteristics of Bacillus subtilis strainDSM
 32560. 4. The composition of claim 1, comprising Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 and/or a strain having all the identifying characteristics ofBacillus amyloliquefaciens strain DSM
 32561. 5. The composition of claim1, comprising Bacillus pumilus strain O72NR7 having deposit accessionnumber DSM 32563 and/or a strain having all the identifyingcharacteristics of Bacillus pumilus strain DSM
 32563. 6. The compositionof claim 1, comprising at least 10⁴ colony forming units of said one ormore Bacillus strains per gram of said composition.
 7. The compositionof claim 1, comprising about 10⁶ to about 10¹² colony forming units ofsaid one or more Bacillus strains per gram of said composition.
 8. Thecomposition of claim 1, comprising about 10⁷ to about 10¹¹ colonyforming units of said one or more Bacillus strains per gram of saidcomposition.
 9. A method comprising mixing the composition of claim 1with one or more feed ingredients to produce an animal feed.
 10. Themethod of claim 9, wherein the composition of claim 1 is mixed with saidone or more feed ingredients in an amount sufficient to produce ananimal feed comprising about 1 x 10¹² colony forming units of said oneor more Bacillus strains per ton of animal feed.
 11. An animal feedcomprising the composition of claim
 1. 12. The animal feed of claim 11,further comprising one or more vitamins, minerals, enzymes, amino acids,preservatives and/or antibiotics.
 13. The animal feed of claim 11,comprising about 1 x 10¹² colony forming units of said one or moreBacillus strains per ton of animal feed.
 14. An animal feed comprisingone or more Bacillus strains selected from the group consisting of:Bacillus licheniformis strain O42AH3 having deposit accession number DSM32559 and strains having all the identifying characteristics of Bacilluslicheniformis strain DSM 32559, Bacillus subtilis strain O52YJ6 havingdeposit accession number DSM 32560 and strains having all theidentifying characteristics of Bacillus subtilis strain DSM 32560,Bacillus amyloliquefaciens strain O52YYT having deposit accession numberDSM 32561 and strains having all the identifying characteristics ofBacillus amyloliquefaciens strain DSM 32561, and Bacillus pumilus strainO72NR7 having deposit accession number DSM 32563 and strains having allthe identifying characteristics of Bacillus pumilus strain DSM 32563.15. The animal feed of claim 14, comprising Bacillus licheniformisstrain O42AH3 having deposit accession number DSM 32559 and/or a strainhaving all the identifying characteristics of Bacillus licheniformisstrain DSM
 32559. 16. The animal feed of claim 14, comprising Bacillussubtilis strain O52YJ6 having deposit accession number DSM 32560 and/ora strain having all the identifying characteristics of Bacillus subtilisstrain DSM
 32560. 17. The animal feed of claim 14, comprising Bacillusamyloliquefaciens strain O52YYT having deposit accession number DSM32561 and/or a strain having all the identifying characteristics ofBacillus amyloliquefaciens strain DSM
 32561. 18. The animal feed ofclaim 14, comprising Bacillus pumilus strain O72NR7 having depositaccession number DSM 32563 and/or a strain having all the identifyingcharacteristics of Bacillus pumilus strain DSM
 32563. 19. The animalfeed of claim 14, further comprising one or more vitamins, minerals,enzymes, amino acids, preservatives and/or antibiotics.
 20. The animalfeed of claim 14, comprising about 1 × 10¹² colony forming units of saidone or more Bacillus strains per ton of said animal feed.